Composite Nanoparticle Structures for Chemical and Biological Sensing

ABSTRACT

Described herein is a nanoparticle that enhances the interaction of the nanoparticle and/or a molecule/material deposited on the surface of the nanoparticle with light, comprising a pair of stacked metallic disks separated by a non-metallic spacer, wherein: (a) the dimensions of the disks and spacer are smaller than the wavelength of the light; and (b) the nanoparticle enhance the light interaction at least three times greater than that an individual metallic disk. Methods for making the nanoparticle and methods for using the nanoparticle in a variety of assays are also provided.

CROSS-REFERENCING

This application is a continuation-in-part of U.S. application Ser. No.13/838,600, filed Mar. 15, 2013 (NSNR-003), which application claims thebenefit of U.S. provisional application Ser. No. 61/622,226 filed onApr. 10, 2012, and is a continuation-in-part of U.S. patent applicationSer. No. 13/699,270, filed on Jun. 13, 2013, which application is a §371filing of US2011/037455, filed on May 20, 2011, and claims the benefitof U.S. provisional application Ser. No. 61/347,178, filed on May 21,2010;

this application is also a continuation-in-part of U.S. application Ser.No. 13/699,270, filed Jun. 13, 2013 (NSNR-001), which application is a§371 filing of international application serial no. US2011/037455, filedon May 20, 2011, which application claims the benefit of U.S.Provisional Patent Application Ser. No. 61/347,178 filed on May 21,2010; and

this application is also claims the benefit of: provisional applicationSer. No. 61/801,424, filed Mar. 15, 2013 (NSNR-004PRV), provisionalapplication Ser. No. 61/801,096, filed Mar. 15, 2013 (NSNR-005PRV),provisional application Ser. No. 61/800,915, filed Mar. 15, 2013(NSNR-006PRV), provisional application Ser. No. 61/793,092, filed Mar.15, 2013 (NSNR-008PRV), provisional Application Ser. No. 61/801,933,filed Mar. 15, 2013 (NSNR-009PRV), provisional Application Ser. No.61/794,317, filed Mar. 15, 2013 (NSNR-010PRV), provisional applicationSer. No. 61/802,020, filed Mar. 15, 2013 (NSNR-011PRV) and provisionalapplication Ser. No. 61/802,223, filed Mar. 15, 2013 (NSNR-012PRV), allof which applications are incorporated by reference herein for allpurposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No.FA9550-08-1-0222 awarded by the Defense Advanced Research Project Agency(DARPA). The government has certain rights in the invention.

BACKGROUND

There is a great need to develop new nanoparticle structures, and newfabrication methods for applications in chemical and biological sensing.The subject nanoparticle structures can greatly enhance (e.g. amplify)optical signals (particularly luminescence (e.g. fluorescence) andSurface Enhanced Raman Scattering (SERS)) disposed on or near to thenanoparticle surface; improve the detection sensitivity of the chemicaland/or biological properties of the molecules disposed on or near thenanoparticles, improve nanoparticle performance in penetratingbiological cells or materials, and in reducing biological toxicities.The subject fabrication methods allow the fabrication of such newnanostructures which are otherwise either impossible or hard tofabricate.

Conventional NPs, limited by their fabrication method (chemicalsynthesis), have simple architecture (spheres, rods, and shells), simpleand smooth surfaces, and simple compositions (either pure dielectric,pure metal, or dielectric enclosed completely by metal (or vice versa)).All of these brought severe drawback to in-vivo diagnosis. The fourmajor drawbacks are: (a) lower plasmonic enhancement than Au clusters orother plasmonic structures made on substrate, (b) large particle sizefor in-vivo diagnosis wavelength (e.g. 300 nm for Au NPs), (c)bio-undegradeable (for metal particles or nanoshells), and (d) similarsurface property in entire surface rather surface location selective.They also have large particle size variations (˜15%). These drawbackslead (i) poor in-vivo performances of low brightness (low enhancement influorescence or SERS), poor in-vivo suitability (slow and low number NPsentering cells), poor biocompability/safety (invasive and particleaccumulation), and poor selectivity

In bio-safe in-vivo plasmonic-based diagnosis and therapeutics, one mostsignificant roadblock is how to satisfy simultaneously two completelyconflicting requirements on nanoparticle size: large (over 50 nm) forbetter therapeutic and diagnostic efficacy (i.e. plasmonic effectivenessand decent blood retention time), and ultra-small (sub10 nm) for lowertoxicity (i.e. rapid clearance from cell/body and hence zeroaccumulation).

Another major roadblock is that conventional nanoparticle fabricationmethods prohibit current plasmonic nanoparticles from having the complexstructures needed for achieving an ultra-high plasmonic enhancement thatis several orders of magnitude higher than the current ones.

For examples, to have decent plasmonic effects and decent bloodretention time, conventional approaches use gold spheres of 300 nmdiameter, nanoshells of 60 nm diameter, and nanorods of 10 diameter and60 nm long. These sizes are much larger than that for quick clear-up incells or bodies, which should be sub-10 nm. Furthermore, for ultra-highplasmonic enhancements, it requires complex particle structures, such asnanogaps and nano-sharp-edges, which are missing in the current NPs,making them several orders of magnitude less plasmonic effective thanwhat we are able to achieve (see section III). Clearly, the current NPscannot simultaneously satisfy the size requirements for plasmoniceffectiveness and bio-safety, because these nanoparticles are notbiodegradable and hence cannot change from large size for plasmoniceffectiveness to small size needed for bio clear-out.

In summary, all previous approaches cannot solve the major roadblocks ofefficacy-safety conflict caused by conflicting particle-sizerequirements nor low plasmonic effects caused by lacking complexity innanostructure.

Therefore, to advance diagnosis (in vitro and in-vivo) and singlebiological cell analysis, we need both new nanoparticle platforms(different architectures and physical principles) and new fabricationmethods, that radically differ from conventional approaches. This issubject of current invention.

SUMMARY

The following brief summary is not intended to include all features andaspects of the present invention, nor does it imply that the inventionmust include all features and aspects discussed in this summary.

The invention is related to nanoparticle structures, fabrication methodsand applications in chemical and biological sensing. The nanoparticlesin the invention has very different structures and materials from theconventional metallic nanoparticles, which allows the new nanoparticlehave many unique properties desired in sensing and diagnostics,including much more effective in enhancing a sensing light signal thanthe conventional metallic nanoparticles while having a size muchsmaller. The small sizes are important to in vivo testing andbio-safety. The unique structures of the nanoparticles cannot be made byconventional synthesis method, but are fabricated by template depositionand exfoliations. The nanoparticles in the invention also canbiodegradable. In particular, the nanoparticle structures can greatlyenhance (e.g. amplify) light absorption, light scattering, and lightradiation, optical signals (particularly luminescence (e.g.fluorescence) and Surface Enhanced Raman Scattering (SERS)) disposed onor near to the nanoparticle surface (the nanoparticle can be insidebiological cell and/or human body); improve the detection sensitivity ofthe chemical and/or biological properties of the molecules disposed onor near the nanoparticles, improve nanoparticle performance inpenetrating biological cells or materials, and in reducing biologicaltoxicities. The subject fabrication methods allow the fabrication ofsuch new nanostructures which are otherwise either impossible or hard tofabricate. The functionalized nanoparticles can be used as biologicaland chemical assay for detection of biological and chemical markers(also termed “analytes”), such as proteins, DNAs, RNAs, and otherorganic and inorganic molecules, in single cells, tissue, and in-vivofor human and animals.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way. Some of thedrawings are not in scale.

FIG. 1 Schematic of Stacked-nanoparticles (S-NPs): (a) a pair ofmetal-disks separated by a single dielectric disk; (b) S-NP with amagnetic disk on top; (c) S-NP with five disks; (d) 4 S-NPs glued byanother dielectric material into a single particle.

FIG. 2 Schematic of (a) DS-NPs: S-NP with metal nano-dots self-assembledat the side wall, and (b) ES-NP (enhanced S-NP) with two non-metallicdisks 460, each of them covers the exterior surface of the metallicdisks of a S-NP.

FIG. 3 Biodegradation of stacked-nanoparticles (S-NPs).

FIG. 4 Schematics of coating a molecular adhesion layer and then thecapture agent.

FIG. 5 schematically illustrates an exemplary antibody detection assay.

FIG. 6 schematically illustrates an exemplary nucleic acid detectionassay.

FIG. 7 schematically illustrates another embodiment nucleic aciddetection assay.

FIG. 8 shows a flow chart of Nano-PrinTED (nanoprint by templatedexfoliatable deposition) and Dip-print of biodegradable dielectrics.Nano-PrinTED comprises three key steps: (i) have a template withnanostructured protrusions or hollows (e.g. dense nanopillar array on 4″wafer), (ii) deposit a release layer (optional) and then multiple-layercomposite materials to form nanoparticles on the pedestals of thetemplate's protrusions or inside the hollows, and (iii) exfoliate thenanoparticles from the template by transfer printing or lift off.Dip-print is for patterning biodegradable dielectrics, since suchmaterials cannot be thermally evaporated. Dip-print first puts a thinlayer of liquid polymer precursors with proper viscosity on a plate(termed “material transfer plate”); and then presses the template usedin Nano-PrinTED against the material transfer plate, picking up thepolymer precursors only at the top of the pillars of the template.Afterwards the polymer precursors will be cured to form a solid polymer.The dip-printed polymers will be used for the dielectrics between thestacked layers and for the dielectrics that glue different columns intoa single particle (Note different viscosity liquid will be useddepending upon the gap size between the columns).

FIG. 9 Nano-PrinTED (nanoprint by templated exfoliateable deposition)—anew nanoparticle fabrication technology. Schematics of (a) Pillartemplate fabricated by lithography (e.g. NIL); (b) multiple depositionand self-assembly to form D2-particles; (c) transfer-print S-NPs toanother substrate; and put in solution.

FIG. 10 Nano-PrinTED (nanoprint by templated exfoliateabledeposition)—method 2. Schematics of (a) hole template (polymer)fabricated by lithography (e.g. NIL); (b) multiple deposition andself-assembly to form D2-particles inside the holes; (c) lift off thepolymer (including the top stacked plane) around S-NPs; and put insolution.

FIG. 11 Dip-print of biodegradable dielectrics. Schematics of (a) pillartemplate fabricated by lithography (e.g. NIL); (b) deposition thin metaldisks on top of the pillars; (c) Puts a thin layer of liquid polymerprecursors with proper viscosity on a plate (termed “material transferplate”); and then presses the template used in Nano-PrinTED against thematerial transfer plate, (d) picking up the polymer precursors only atthe top of the pillars of the template. (e) add another deposition stepto form top metal disk if needed and (f) put S-NPs into solutions.

FIG. 12 Dip-print of biodegradable dielectrics that “glue” columns. Thesame as dip printing the dielectric spacer, except that the viscosity ofpolymer precursors may be changed to make the polymer precursor fill thegaps between the columns. (a) 4 close pillar template with 4 S-NPs ontop. (b) pick up the polymer precursors at the top of the S-NPs on thetemplate, fill the gaps in between and glue the 4 S-NPs. (c) put intosolutions.

FIG. 13 Scanning electron microscopy (SEMs) of (a) double-metal-disk andsingle dielectric (D-particle); (b) triple-metal (or magnetic)dielectric-nanoparticle (TS-NP); (c) D-particle after the“self-perfection by liquefaction” (SPEL) to change the shape of 2 metaldisks; (d) D-particles array on the substrate after the templatelift-off.

FIG. 14 Scanning electron microscopy (SEMs) of (a) D-particles array onthe substrate after the transfer printing. (b) D-particles exfoliatedinto solution.

FIG. 15 Nano-PrinTED (nanoprint by templated exfoliateable deposition)—anew nanoparticle fabrication technology. Top row: Schematic. And bottomrow: scanning electron microscope (SEM) of experimental results. (a)Pillar template fabricated by lithography (e.g. NIL); (b) Multipledeposition and self-assembly to form D2-particles; (c) transfer-printDPs to another substrate; (d) put in solution. (e-h), SEM images.

FIG. 16 Nano-PrinTED and Dip-print have far better precision incontrolling the NP structure dimensions (including the size and shape ofeach individual components, their spacing, and final particle). (a) SEMpicture of D2-P before release and (b) Measured size distribution.Measured size variation of D2-particle fabricated by Nano-PrinTED (<5%)is 3 fold less than AuNP manufactured by chemical synthesis (>15%).

FIG. 17 Measurements of extinction spectrum of D2-particles with SiO₂layer thickness from 5 nm to 30 nm and constant Au layer thicknesses of20 nm. Plasmonic resonant peak wavelengths redshift with increasing SiO₂layer thickness.

FIG. 18 Simulation of the size of nanoparticles with differentarchitectures required for the same resonant wavelength at 800 nm. Itclearly shows that S-NP has much smaller particle sized thanconventional metallic sphere and disks for a given resonant wavelength.

FIG. 19 (a) Measured Surface Enhanced Raman Spectroscopy (SERS) signalof BPE, and (b) fluorescence signal of IR-800 dye with singleD2-particle and gold nanoparticle. A single D2-particle has aSERS/Fluorescence enhancement over 100/30 fold higher than a single goldnanoparticle of similar diameter.

Corresponding reference numerals indicate corresponding parts throughoutthe several figures of the drawings. It is to be understood that thedrawings are for illustrating the concepts set forth in the presentdisclosure and are not to scale.

Before any embodiments of the invention are explained in detail, it isto be understood that the invention is not limited in its application tothe details of construction and the arrangement of components set forthin the following description or illustrated in the drawings.

DEFINITIONS

Before describing exemplary embodiments in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used in the description.

The term “molecular adhesion layer” refers to a layer or multilayer ofmolecules of defined thickness that comprises an inner surface that isattached to the S-NP and an outer (exterior) surface can be bound tocapture agents.

The term “capture agent-reactive group” refers to a moiety of chemicalfunction in a molecule that is reactive with capture agents, i.e., canreact with a moiety (e.g., a hydroxyl, sulfhydryl, carboxy or aminegroup) in a capture agent to produce a stable strong, e.g., covalentbond.

The term “capture agent” as used herein refers to an agent that binds toa target analyte through an interaction that is sufficient to permit theagent to bind and concentrate the target molecule from a heterogeneousmixture of different molecules. The binding interaction is typicallymediated by an affinity region of the capture agent. Typical captureagents include any moiety that can specifically bind to a targetanalyte. Certain capture agents specifically bind a target molecule witha dissociation constant (K_(D)) of less than about 10⁻⁶ M (e.g., lessthan about 10⁻⁷ M, less than about 10⁻⁸ M, less than about 10⁻⁹ M, lessthan about 10⁻¹⁹ M, less than about 10⁻¹¹ M, less than about 10⁻¹² M, toas low as 10⁻¹⁶ M) without significantly binding to other molecules.Exemplary capture agents include proteins (e.g., antibodies), andnucleic acids (e.g., oligonucleotides, DNA, RNA including aptamers).

The term “nanosensor” refers to a nanoparticle that is functionalizewith a capture agent.

The terms “specific binding” and “selective binding” refer to theability of a capture agent to preferentially bind to a particular targetmolecule that is present in a heterogeneous mixture of different targetmolecule. A specific or selective binding interaction will discriminatebetween desirable (e.g., active) and undesirable (e.g., inactive) targetmolecules in a sample, typically more than about 10 to 100-fold or more(e.g., more than about 1000- or 10,000-fold).

The term “protein” refers to a polymeric form of amino acids of anylength, i.e. greater than 2 amino acids, greater than about 5 aminoacids, greater than about 10 amino acids, greater than about 20 aminoacids, greater than about 50 amino acids, greater than about 100 aminoacids, greater than about 200 amino acids, greater than about 500 aminoacids, greater than about 1000 amino acids, greater than about 2000amino acids, usually not greater than about 10,000 amino acids, whichcan include coded and non-coded amino acids, chemically or biochemicallymodified or derivatized amino acids, and polypeptides having modifiedpeptide backbones. The term includes fusion proteins, including, but notlimited to, fusion proteins with a heterologous amino acid sequence,fusions with heterologous and homologous leader sequences, with orwithout N-terminal methionine residues; immunologically tagged proteins;fusion proteins with detectable fusion partners, e.g., fusion proteinsincluding as a fusion partner a fluorescent protein, β-galactosidase,luciferase, etc.; and the like. Also included by these terms arepolypeptides that are post-translationally modified in a cell, e.g.,glycosylated, cleaved, secreted, prenylated, carboxylated,phosphorylated, etc., and polypeptides with secondary or tertiarystructure, and polypeptides that are strongly bound, e.g., covalently ornon-covalently, to other moieties, e.g., other polypeptides, atoms,cofactors, etc.

The term “antibody” is intended to refer to an immunoglobulin or anyfragment thereof, including single chain antibodies that are capable ofantigen binding and phage display antibodies).

The term “nucleic acid” and “polynucleotide” are used interchangeablyherein to describe a polymer of any length composed of nucleotides,e.g., deoxyribonucleotides or ribonucleotides, or compounds producedsynthetically (e.g., PNA as described in U.S. Pat. No. 5,948,902 and thereferences cited therein) which can hybridize with naturally occurringnucleic acids in a sequence specific manner analogous to that of twonaturally occurring nucleic acids, e.g., can participate in Watson-Crickbase pairing interactions.

The term “complementary” as used herein refers to a nucleotide sequencethat base-pairs by hydrogen bonds to a target nucleic acid of interest.In the canonical Watson-Crick base pairing, adenine (A) forms a basepair with thymine (T), as does guanine (G) with cytosine (C) in DNA. InRNA, thymine is replaced by uracil (U). As such, A is complementary to Tand G is complementary to C. Typically, “complementary” refers to anucleotide sequence that is fully complementary to a target of interestsuch that every nucleotide in the sequence is complementary to everynucleotide in the target nucleic acid in the corresponding positions.When a nucleotide sequence is not fully complementary (100%complementary) to a non-target sequence but still may base pair to thenon-target sequence due to complementarity of certain stretches ofnucleotide sequence to the non-target sequence, percent complementarilymay be calculated to assess the possibility of a non-specific(off-target) binding. In general, a complementary of 50% or less doesnot lead to non-specific binding. In addition, a complementary of 70% orless may not lead to non-specific binding under stringent hybridizationconditions.

The terms “ribonucleic acid” and “RNA” as used herein mean a polymercomposed of ribonucleotides.

The terms “deoxyribonucleic acid” and “DNA” as used herein mean apolymer composed of deoxyribonucleotides.

The term “oligonucleotide” as used herein denotes single strandednucleotide multimers of from about 10 to 200 nucleotides and up to 300nucleotides in length, or longer, e.g., up to 500 nt in length orlonger. Oligonucleotides may be synthetic and, in certain embodiments,are less than 300 nucleotides in length.

The term “attaching” as used herein refers to the strong, e.g, covalentor non-covalent, bond joining of one molecule to another.

The term “surface attached” as used herein refers to a molecule that isstrongly attached to a surface.

The term “sample” as used herein relates to a material or mixture ofmaterials containing one or more analytes of interest. In particularembodiments, the sample may be obtained from a biological sample such ascells, tissues, bodily fluids, and stool. Bodily fluids of interestinclude but are not limited to, amniotic fluid, aqueous humour, vitreoushumour, blood (e.g., whole blood, fractionated blood, plasma, serum,etc.), breast milk, cerebrospinal fluid (CSF), cerumen (earwax), chyle,chime, endolymph, perilymph, feces, gastric acid, gastric juice, lymph,mucus (including nasal drainage and phlegm), pericardial fluid,peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil),semen, sputum, sweat, synovial fluid, tears, vomit, urine and exhaledcondensate. In particular embodiments, a sample may be obtained from asubject, e.g., a human, and it may be processed prior to use in thesubject assay. For example, prior to analysis, the protein/nucleic acidmay be extracted from a tissue sample prior to use, methods for whichare known. In particular embodiments, the sample may be a clinicalsample, e.g., a sample collected from a patient.

The term “analyte” refers to a molecule (e.g., a protein, nucleic acid,or other molecule) that can be bound by a capture agent and detected.

The term “assaying” refers to testing a sample to detect the presenceand/or abundance of an analyte.

As used herein, the terms “determining,” “measuring,” and “assessing,”and “assaying” are used interchangeably and include both quantitativeand qualitative determinations.

As used herein, the term “light-emitting label” refers to a label thatcan emit light when under an external excitation. This can beluminescence. Fluorescent labels (which include dye molecules or quantumdots), and luminescent labels (e.g., electro- or chemi-luminescentlabels) are types of light-emitting label. The external excitation islight (photons) for fluorescence, electrical current forelectroluminescence and chemical reaction for chemi-luminscence. Anexternal excitation can be a combination of the above.

The phrase “labeled analyte” refers to an analyte that is detectablylabeled with a light emitting label such that the analyte can bedetected by assessing the presence of the label. A labeled analyte maybe labeled directly (i.e., the analyte itself may be directly conjugatedto a label, e.g., via a strong bond, e.g., a covalent or non-covalentbond), or a labeled analyte may be labeled indirectly (i.e., the analyteis bound by a secondary capture agent that is directly labeled).

The term “hybridization” refers to the specific binding of a nucleicacid to a complementary nucleic acid via Watson-Crick base pairing.Accordingly, the term “in situ hybridization” refers to specific bindingof a nucleic acid to a metaphase or interphase chromosome.

The terms “hybridizing” and “binding”, with respect to nucleic acids,are used interchangeably.

The term “capture agent/analyte complex” is a complex that results fromthe specific binding of a capture agent with an analyte. A capture agentand an analyte for the capture agent will usually specifically bind toeach other under “specific binding conditions” or “conditions suitablefor specific binding”, where such conditions are those conditions (interms of salt concentration, pH, detergent, protein concentration,temperature, etc.) which allow for binding to occur between captureagents and analytes to bind in solution. Such conditions, particularlywith respect to antibodies and their antigens and nucleic acidhybridization are well known in the art (see, e.g., Harlow and Lane(Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1989) and Ausubel, et al, Short Protocols inMolecular Biology, 5th ed., Wiley & Sons, 2002).

The term “specific binding conditions” as used herein refers toconditions that produce nucleic acid duplexes or protein/protein (e.g.,antibody/antigen) complexes that contain pairs of molecules thatspecifically bind to one another, while, at the same time, disfavor tothe formation of complexes between molecules that do not specificallybind to one another. Specific binding conditions are the summation orcombination (totality) of both hybridization and wash conditions, andmay include a wash and blocking steps, if necessary.

For nucleic acid hybridization, specific binding conditions can beachieved by incubation at 42° C. in a solution: 50% formamide, 5×SSC(150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC atabout 65° C.

For binding of an antibody to an antigen, specific binding conditionscan be achieved by blocking a substrate containing antibodies inblocking solution (e.g., PBS with 3% BSA or non-fat milk), followed byincubation with a sample containing analytes in diluted blocking buffer.After this incubation, the substrate is washed in washing solution (e.g.PBS+TWEEN 20) and incubated with a secondary capture antibody (detectionantibody, which recognizes a second site in the antigen). The secondarycapture antibody may conjugated with an optical detectable label, e.g.,a fluorophore such as IRDye800CW, Alexa 790, Dylight 800. After anotherwash, the presence of the bound secondary capture antibody may bedetected. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected and toreduce the background noise.

The term “a secondary capture agent” which can also be referred to as a“detection agent” refers a group of biomolecules or chemical compoundsthat have highly specific affinity to the antigen. The secondary captureagent can be strongly linked to an optical detectable label, e.g.,enzyme, fluorescence label, or can itself be detected by anotherdetection agent that is linked to an optical detectable label throughbioconjugatio (Hermanson, “Bioconjugate Techniques” Academic Press, 2ndEd., 2008).

The term “biotin moiety” refers to an affinity agent that includesbiotin or a biotin analogue such as desthiobiotin, oxybiotin,2′-iminobiotin, diaminobiotin, biotin sulfoxide, biocytin, etc. Biotinmoieties bind to streptavidin with an affinity of at least 10-8M. Abiotin affinity agent may also include a linker, e.g., —LC-biotin,—LC-LC-Biotin, —SLC-Biotin or —PEGn-Biotin where n is 3-12.

The term “streptavidin” refers to both streptavidin and avidin, as wellas any variants thereof that bind to biotin with high affinity.

The term “marker” refers to an analyte whose presence or abundance in abiological sample is correlated with a disease or condition.

The term “bond” includes covalent and non-covalent bonds, includinghydrogen bonds, ionic bonds and bonds produced by van der Waal forces.

The term “amplify” refers to an increase in the magnitude of a signal,e.g., at least a 10-fold increase, at least a 100-fold increase at leasta 1,000-fold increase, at least a 10,000-fold increase, or at least a100,000-fold increase in a signal.

The term “local” refers to “at a location”,

Other specific binding conditions are known in the art and may also beemployed herein.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise, e.g., when the word “single” isused. For example, reference to “an analyte” includes a single analyteand multiple analytes, reference to “a capture agent” includes a singlecapture agent and multiple capture agents, and reference to “a detectionagent” includes a single detection agent and multiple detection agents.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The following detailed description illustrates some embodiments of theinvention by way of example and not by way of limitation.

The invention is related to nanoparticle structures, fabrication methodsand applications in chemical and biological sensing. The nanoparticlesin the invention has very different structures and materials from theconventional metallic nanoparticles, which allow the new nanoparticlehaving many unique properties desired in sensing and diagnostics,including much more effective in enhancing a sensing light signal thanthe conventional metallic nanoparticles while having a size muchsmaller. The small sizes are important to in vivo testing andbio-safety. The unique structures of the nanoparticles cannot be made byconventional synthesis method, but are fabricated by template depositionand exfoliations. The nanoparticles in the invention also can bebiodegradable. In particular, the nanoparticle structures can greatlyenhance (e.g. amplify) light absorption, light scattering, and lightradiation, optical signals (particularly luminescence (e.g.fluorescence) and Surface Enhanced Raman Scattering (SERS)) disposed onor near to the nanoparticle surface (the nanoparticle can be insidebiological cell and/or human body); improve the detection sensitivity ofthe chemical and/or biological properties of the molecules disposed onor near the nanoparticles (e.g. proteins, DNAs, RNAs, and other organicand inorganic molecules), improve nanoparticle performance inpenetrating biological cells or materials, and in reducing biologicaltoxicities. The subject fabrication methods allow the fabrication ofsuch new nanostructures which are otherwise either impossible or hard tofabricate.

The invention covers four areas: (1) nanoparticle structures, (2)fabrication methods, (3) surface functioning, and (4) applications inchemical and biological sensing.

Certain physical principles and certain materials, dimensions gaps usedin the invention are similar to “disk-coupled dots-on-pillar antennaarray” (D2PA) which is on a solid support (as described in WO2012/024006and WO2013154770 which are incorporated by reference).

Nanoparticle Structures and Materials Basic Structure

In one embodiment of the invention, as illustrated in FIG. 1, ananoparticle 100, termed stacked-nanoparticle (S-NP), that enhances theinteraction of the nanoparticle and/or a molecule/materials deposited onthe surface of the nanoparticle with light, comprises at least a pair ofstacked metallic disks 110 and 120, separated by a non-metallic spacer130, wherein (a) the dimensions of the disks and spacer are smaller thanthe wavelength of the light; and (b) the nanoparticle enhances the lightinteraction at least three times greater than that of each said metallicdisk. The metallic disks 110 and 120 can be made of the same ordifferent materials and have the same or different thickness.Furthermore, a thin adhesion layer may be between two adjacent disks.

The non-metallic spacer 130 can be biodegradable, namely, it can bedissolved in a bio-environment. As illustrated in FIG. 3, when thespacer is degraded, a S-NP breaks into small pieces. Breaking up intosmall pieces has advantages in in vivo application; small pieces cancome out biological cells and human body much faster than large pieces,hence avoiding accumulation. Since the space is not completely enclosedby the metallic disks (non-biodegradable), fluid can access the spacerfrom the side of the spacer. The biodegradation time can be controlledby the biodegradable material and their dimensions.

The light interaction include light absorption, light scattering, lightradiation, Raman scattering, chromaticity, and luminescence thatincludes fluorescence, electroluminescence, chemiluminescence, andelectrochemiluminescence.

A preferred wavelength range for the light that can be enhanced by theS-NP is about from 20 nm to 10 micrometer. Another preferred thewavelength range is from 300 nm to 4000 nm. For in vivo applications, apreferred wavelength range (window) for a good light penetration inbiological tissue is about from 630 nm to 1316 nm.

The molecule/materials deposited on the S-NP include themolecules/materials to be sensed, such as the analytes and/or theirlabels including proteins, DNAs, RNAs, and other organic and inorganicmolecules in single cells, tissue, and in-vivo for human and animals.

The “metallic” in this invention means that for a given light wavelengththe electrons in the materials can generate plasmons. For example, thegold has a plasmon wavelength about 560 nm; for the wavelength longerthan 560 nm, the gold behavior like “metallic”, for the wavelengthsignificantly shorter than 560 nm, the gold behavior like a non-metallicto the light.

The sensing property includes the sensing signal intensity, sensingsignal spectrum, limit of detection, detection dynamic range, and signalvariation reduction (smaller error bar) of the sensing. The sensingincludes the detection of the existence, quantification of theconcentration, and determination of the states of the targeted analyte.The analyte includes proteins, DNAs, RNAs, and other organic andinorganic molecules. The invention can be used in the sensing in vitro,or in vivo.

The key reason for S-NP superior to the conventional NPs in enhancementwith smaller size is due to different physics. Conventional NPs withmetal all around follows the parabolic function or the dispersionrelation

${{\frac{k_{x}^{2}}{ɛ_{1}} + \frac{k_{y}^{2}}{ɛ_{2}}} = \frac{\omega^{2}}{c^{2}}},$

where ∈₁ and ∈₂ are dielectric constants (permittivities) in twoorthogonal directions and for conventional NPs, they both have the samesign. Hence, their isofrequency curve is elliptical, which leads tobounded wavevectors, k, and relatively long wavelength. But for S-NPwith bipolar-permittivity (let ∈₁=∈_(p) and ∈₂=−∈_(v), having oppositesign, while ∈_(p) and ∈_(v) are positive values), the equation becomes

${{\frac{k_{v}^{2}}{ɛ_{p}} - \frac{k_{p}^{2}}{ɛ_{v}}} = \frac{\omega^{2}}{c^{2}}},$

a hyperbolic curve, which means that wavevector, k, in both directionsis unbounded, and can be very large. This allows a very short wavelengthinside the particle (outside the particle the wavelength is still 800nm), and consequently a very small particle size for a large opticalsignal. Furthermore, a pair of disks forms a resonant cavity for thelight.

As shown in FIG. 4, when the surface of the S-NP 100 is functionalized,it becomes a nanosensor 200 for sensing an analyte in biological andchemical detection. The surface functionalization can be many ways asdiscussed later, including attaching the capture agents that selectivelybond to a targeted analyte. The analytes include proteins, peptides,DNA, RNA, nucleic acid, small molecules, cells, nanoparticles withdifferent shapes. The targeted analyte can be either in a solution or inair or gas phase. The sensing includes light absorption, lightscattering, light radiation, Raman scattering, chromaticity,luminescence that includes fluorescence, electroluminescence,chemiluminescence, and electrochemiluminescence. The sensing propertyincludes the sensing signal intensity, sensing signal spectrum, limit ofdetection, detection dynamic range, and signal variation reduction(smaller error bar) of the sensing. The sensing includes the detectionof the existence, quantification of the concentration, and determinationof the states of the targeted analyte. The invention can be used in thesensing in vitro, or in vivo.

NP Structure Variations and Improvements

Dots-on-Sidewall S-NP (DS-SP).

One embodiment of S-NP (FIG. 1 b), termed “dots-on-sidewall S-NP”(DS-SP) 400 comprises metallic nanodots 440 on the sidewall of thespacer and/or the metallic disks, which can have a higher light signalenhancement than the S-NP without them. For 800 nm wavelength, thediameter of the nanodots is about 3 to 15 nm.

Magnetic/Magnetizable S-NP (MS-NS).

S-NP can be made to be magnetic/magnetizable, so that they can beattracted to a magnet, and is termed MS-NP. One embodiment of MS-NPcomprises a S-NP having at least one magnetic/magnetizamble disk 140stacked on a normal S-NP, as shown in FIG. 1 b.

Enhanced S-NP (ES-NP).

The S-NP light signal enhancement can be further increased. For a S-NP,the light signal enhancement on its surface is not uniform: the regionswith sharp (i.e. small curvature) edges of metallic materials and thesmall gaps between two metallic materials are the high enhancementregions, while the other regions are low enhancement regions (e.g. theflat exterior surface of the metallic disk). The high enhancement regionmeans that a molecule or a material attached to that region will haveits light signal amplified more than that of attaching to a lowenhancement region. For example, a fluorophore (e.g. a fluorescencemolecule) attached to the high enhancement region will have a higherfluorescence signal under a light excitation than that to attached a lowenhancement region.

One embodiment of the invention is to selectively mask the lowenhancement regions from a molecular binding and selectively attach themolecules that its light signal will be amplified (e.g. the captureagent that binds an analyte with a light label) to the high enhancementregions. One way to achieve this is to add two non-metallic disks asmasking disks, one on each side of the S-NP, to mask the exteriorsurface of the metallic disks of a S-NP from the attachment of amolecule, as illustrated 150 and 160 in FIG. 1 c, and 460 in FIG. 2.Such particle 400 is termed ES-NP (enhanced S-NP), which has five disks(2 metallic disks and 3 non-metallic disks). For example, a 3 nm thickof SiO₂ disk can be used as the masking disks and the molecules are DSU,which only attach to the metal. Of course, one chose to mask one-side ofdisk or both size. The masking disks may use different thickness anddifferent materials. For some applications, a part of disk edges arealso masked. Some other details have be disclosed in PCT/US14/29979,filed on Mar. 15, 2014, and 61/801,424, filed on Mar. 15, 2013, whichare incorporated by reference herein.

S-NP with More Stacked Metallic Disks.

In certain embodiments, S-NP has more than three disks; it can have 4,5, 6, or more disks as much as required in sensing. The metallic disksalso can be more a pair.

Bundled S-NPs (BS-NP) (S-NP with Multi-Columns).

One embodiment of S-NP is to pack several S-NPs together into one largerparticle (a bundle) using a biodegradable dielectric material glue (FIG.1.D). The reason is that such bundle allows the bundle having the sameor similar light signal enhancement as a single S-NP with the same size,but after biodegradable, the bundle has much smaller pieces and henceeasy to be cleared from biological cells and human body.

Disks Shape and Dimension

The lateral shapes of the disks of S-NP can be selected from the shapeof round, square, rectangle, polygon, elliptical, elongated bar,polygon, other similar shapes or combinations thereof. In general, eachdisk can have different lateral shapes and dimensions from the others.In certain cases, as discussed later, one way to fabricate the S-NP isby using a template, deposition, exfoliation; such fabrication leads tosimilar lateral shape and dimensions for all disks in a given S-NP. Butby using different templates each S-NP can have may different lateralshapes.

The shape of the top and bottom surface of the disks can be different,and can be flat, but also can be bulged, or a half-sphere. An example ofthe fabricated S-NP for 800 nm light wavelength are given in FIGS. 13,14, and 15.

Dimensions for the metallic disks of S-NP and the spacer should be lessthan the wavelength of the light that the S-NP enhances. For a givenwavelength, the light enhancement depends on the S-NP size and aresonant peak (vs. the size). The spacer between the pair of metallicdisk has a thickness of 0.3 nm to 50 nm. This spacer's thickness has animportant role in determine gap. In general smaller the gap is better,but a small gap also changes the resonant wavelength.

The disk diameters are often decided by balancing the light signalenhancement and the other requirements in in vivo application. For anexample, to have an easy bio-cell and bio-material penetration and exitand bio-safety, the particle size should be as small as possible, but ifthe particle size is too small, it reduces the light enhancement factor.One embody of the invention is the S-NP that optimize both requirements.

As an example of DS-NP for 800 nm wavelength, the disks have a roundshape of diameter from 30 nm to 100 nm, the top metallic disk thicknessis from 5 nm to 30 nm, the spacer thickness is from 2 to 30 nm, and thebottom metallic disk thickness is from 5 nm to 30 nm, the self-assembleddots diameter is 5 nm to 15 nm, the magnetic disk thickness is from 5 to30 nm, and the adhesion layers between the disks are titanium or Cr of athickness from 0.5 nm-1 nm. Examples of the fabricated disks are shownin FIG. 13-16.

In a preferred DS-NP structure with light resonance absorption around800 nm wavelength, the disks have a round shape of diameter of 70 nm,the top metallic (gold) disk thickness is 15 nm, the spacer (silicondioxide) thickness is 20 nm, and the bottom metallic (gold) diskthickness is 15 nm, the self-assembled gold dots diameter is around 10nm, and the adhesion layers between the disks are titanium of athickness of 0.5 nm.

Metallic Materials

The materials used for the metal components (e.g. disk and dots) inS-NPs are chosen from materials that are metallic in the working photonwavelength. For examples, the metallic materials can be selected fromgold, copper, silver, aluminum, their mixture, alloys, and multilayersin visible light ranges and longer wavelength, and certain metal oxides(as ITO, zinc oxide), for near or mid infrared wavelength or longerwavelength, or semiconductor (as silicon or gallium arsenide) forcertain wavelength range. One can use a single material or a combinationof them.

Materials for Non-Metallic Spacer.

The materials for the non-metallic spacer and non-metallic maskinglayers in S-NPs are chosen from dielectric materials and/orsemiconductors. The material can be bio-degradable ornon-bio-degradable. One important condition in selecting these materialsis their effects on the light enhancement of S-NP. In many embodiments,such enhancement should be as high as allowed.

The dielectric materials can be inorganic or organic either in crystal,polycrystalline, amorphous, or hetero-mixture, and combination of one ormore thereof depends on the applications. For examples, inorganicdielectric components can be selected from silicon dioxide, diamond,graphite, titanium dioxide, other certain metal oxides, and inorganiccompounds in light wavelength range smaller than their energy bandgap.Organic dielectric components can be selected from polymers asbiodegradable polymers (list before) for certain applications, otherpolymer as biopolymer (e.g. polynucleotides, cellulose), copolymer (e.g.styrene-isoprene-styrene), conductive polymer (e.g. poly(p-phenylenevinylene)), fluoropolymer, polyterpene, phenolic resin, polyanhydrides,polyester, polyolefin, rubber, superabsorbent polymer, vinyl polymer,etc.; or from small molecules, e.g. fullerene derivative, benzenederivatives, etc. The semiconductor materials can be inorganic ororganic either in crystal, polycrystalline, amorphous, orhetero-mixture, and combination of one or more thereof depends on theapplications. One can use a single material or a combination of them.

Biodegradable Materials

The biodegradable polymers for S-NPs are a specific type of polymersthat is stable and durable enough for use in their intended applicationsand easily breaks down (to form gases, salts, or biomass) after itsdegradation.

These biodegradable polymers contains two major types: agro-polymers(derived from biomass, e.g. poly(saccharide)s as the starches in wood,cellulose, chitosan, proteins), and biopolyesters (derived frommicro-organisms or synthetically made from either naturally or syntheticmonomers, e.g. polyhydroxybutyrate and polylactic acids). Most ofbiodegradable polymers consist of ester, amide, or ether bonds. Moreexamples of these biodegradable polymers contains: poly(esters) based onpolylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), andtheir copolymers, poly(hydroxyalkanoate)s of the PHB-PHV class, otherhydrolytically degradable polymers (as polyurethanes, poly(ester amide),poly(ortho esters), polyanhydrides, poly(anhydride-co-imide), pseudopoly(amino acide), poly(alkyl cyanoacrylates), etc.), otherenzymatically degradable polymers (proteins (as collagen, elastion),poly(amino acids), polysaccharides, etc), and other natural polymers.One can use a single material or a combination of them.

Magnetic/Magnetizable Materials

The magnetic/magnetizable materials are those materials that willexperience a magnetic force in a magnetic field. The can be selectedfrom: ferromagnetic (e.g. iron, cobalt, nickel, some of the rare earths(gadolinium, dysprosium), etc.), ferrimagnetic (e.g. iron(II,III) oxide,yttrium iron garnet, cubic ferrites composed of iron oxides and otherelements such as aluminum, cobalt, nickel, manganese and zinc, hexagonalferrites, and pyrrhotite, etc.), superparamagnetic, and other suitablemagnetic materials include oxides, e.g. ferrites, perovskites, chromitesand magnetoplumbites, a rare earth/cobalt alloy or any otherinorganic/organic compound with ferromagnetic/ferrimagnetic properties.One can use a single material or a combination of them.

Some key advantages are: the S-NPs have the right (large) particle sizeand complex shape for high-performance in-vivo plasmonic enhanceddiagnostics and therapeutics, yet biodegradable, afterwards (withcontrolled timing), into sub-10-nm particles with a volume only 10% to1% of original S-NP for quick cell/body clearance. Furthermore, S-NPsoffer a plasmonic enhancement several orders of magnitudes higher thanall current NPs.

To design different biocompatible and biodegradable S-particles that arenot only effective in in-vivo therapeutic and diagnosis but alsobio-safe, we need to control different S-NP's architectures, materials,dimensions and shapes.

1. Particle size requirements for efficacy of plasmonic-based in-vivotherapeutic and diagnosis. Two factors that determine the sizes: (a)effectiveness of plasmonic effects and (b) the blood retention time—thetime needed for sufficient circulation in blood to deliver proper dosageof nanoparticles to the specific sites.

To be plasmonic effective needs two things: first, the nanoparticle sizehas to be in resonance with in coming wavelength; and second, thereshould be small gaps and sharp edges. For the first plasmonicrequirement at the in vivo penetration light wavelength of 800 nm (theentire window is NIR (670 to 890 nm)), for example, conventionalapproaches use gold spheres of 300 nm diameter, gold nanoshells of 60 nmdiameter, and gold nanorods of 10 diameter and 60 nm long for decentplasmonic effects and decent blood retention time. The size smaller thanthose above will make these particles plasmonic extremely ineffective.

For the second plasmonic requirement, the conventional particles do nothave the complex structures (nanogaps and nano-edges), and hence aremuch worse (orders of magnitude worse) than those that have suchstructures, which has been proofed by our experiment with S-particles.

To have sufficient blood retention time, the NP's size should be −50 to200 nm range, and cannot be too small either. Too small particle sizewill make the particle cleared-out from cells and body quickly (seebelow), hence failing to deliver proper dosage, unless largenanoparticle dosage or repeated dosage of nanoparticles areintravenously administered, which will become unsafe and causeimmunogenic response.

2. Particle size requirements for bio-safety. Two nanoparticle sizes arevery important in bio-safety in in-vivo diagnosis: (a)safe-particle-delivery size, which is the nanoparticle size that can besafely delivered to the specific sites without causing immunogenicresponse or any toxic effects, assuming low NP dose and no NPaccumulation, and (b) particle clearance size, where NPs with such sizecan be easily cleared out from cells/body. As shown in FIG. 11, theparticle safe-particle-delivery size has several bands, and the particleclearance size should be <10 nm (<6 nm even better).

3. Conflict in particle size requirements. Clearly, the sizerequirements are conflicting. For conventional plasmonic particles,which cannot change their size once are put in vivo, only way to solvethis size requirement conflict is to compromise both the efficacy andthe bio-safety to pick a particle size in the middle to balanceplasmonic enhancement and the circulation within the body with thenanoparticles' ability to escape from the body.

The exact dimensions of these components depend on the light wavelengthand materials. In some cases particular for the light wavelength of ˜800nm and gold for the metallic materials and silicon dioxide as thedielectrics, the dimensions of the components can be in the range of 4nm to 1500 nm and a thickness of the disks may be from 1 nm to 500 nm,depending on the exact wavelength of the light to be used in sensing.The gaps between components (e.g., the gaps between the metallic disks)may be in the range of 0.5 nm to 200 nm. For many applications, a smallgap (in the range of 5 nm to 50 nm) may be used to enhance the opticalsignals.

This is one of the most challenging issues in plasmonic-based particlein vivo. Previous approaches have very limited success. For example,nanoparticle such as PEG-passivated gold nanoparticles, Nanoshells andgold nanorods. These nanoparticles employs surface coating or exoticshapes to achieve optimum optical resonance while retain a proper size(<100 nm) throughout the needed blood retention time. However, theirability of tuning the optical responses is still limited, thus theoptical field enhancement is still weak. Moreover, these particles havea size much larger than the clearance size and hence will be accumulatedin the body, becoming toxic.

S-NP Fabrication Methods

Nano-PrinTED-1 (Protrusion Template).

As shown in FIGS. 8 and 9, one embodiment of the fabrication method,termed Nano-PrinTED using a protrusion template 1010, comprising threekey steps: (i) have a template with nanostructured protrusions 1010(e.g. dense nanopillar array on 4″ wafer (each pillar of the same orsimilar diameter selected from 5 nm to 100 nm), (ii) deposit a releaselayer (optional) and then deposit multiple-layers of materials 1020needed to form nanoparticles on both the pedestals of the template'sprotrusions (each nanoparticle size is determined by the diameter of thetemplate nanopillar) and inside the trenches, and (iii) exfoliate thenanoparticles 100 from the template.

Solvent Dissolve/Ultra-Sonic Exfoliation.

The template with S-NPs is put inside the container with particularsolvent to dissolve the release layer under the S-NPs. The container canbe put in an ultrasonicator to speed up the exfoliation process. S-NPswill be exfoliated in the solvent.

The exfoliation can be done in several ways: (1) Transfer printingexfoliation: The template with S-NPs is printed onto another substratewith a thin adhesion layer 1030 (e.g. certain polymer thin film). Thelarger adhesion force between the particles and the adhesion layerexfoliate all the S-NPs onto the new substrate. The S-NPs can be furtherreleased into solution by dissolving the adhesion layer on the newsubstrate similar to previous method. (2) Spin-on peel-off exfoliation:A kind of adhesion thin film (e.g. certain polymer) is spinned onto thetemplate with S-NPs. After the curing of the film, the S-NPs are adheredin the adhesion layer, following a peel-off process to exfoliate all theS-NPs. The S-NPs can be further released into solution by dissolving theadhesion film as previous method. And (3) Wash exfoliation: The templatewith S-NPs are tilted above a beaker (or other container), and washedwith certain solution (with spray gun). With the force of turbulentsolution, the S-NPs will be exfoliated from the template and into thesolution.

In certain embodiments, the same metal deposition for 1020 also form thenanodots on the disk sidewall to form S-NP, due to the fact that a thinmetal on the sidewall self-assembled into nanodots, as shown from theexperimental results in FIGS. 13, 14 and 15.

In the deposition (ii), the deposition uses a beamed materials (i.e. thematerial is deposited in one direction not in the other directions) andin the angle substantially normal to the template surface. Due theheight of the pillars, the materials deposited in on the foot of thepillars are not connected to the materials deposited on the top of thepillar (i.e. pillars' pedestals), making the materials deposited on thepedestals forming S-NPs. The exfoliation free these S-NP from thetemplate. The template can be used repeatedly until the trenches betweenthe pillars are filled with material. When that happens, a cleaning stepcan be used to remove the deposited materials, and then the template canbe reused again.

Nano-PrinTED-2 (Concave Template).

As shown in FIG. 10, one embodiment of the fabrication method, termedNano-PrinTED using a concave template, comprising three key steps: (i)have a template with nanostructured wells 1100, (ii) deposit a releaselayer (optional) and then deposit multiple-layers of materials 1120needed to form nanoparticles in the wells of the template's protrusions(each nanoparticle size is determined by the diameter of the well), and(iii) exfoliate the nanoparticles 100 from the template. Due the depthof the wells, the materials deposited on the bottom of the wells are notconnected to the materials deposited on the top surface of the template,making the materials deposited inside well forming S-NPs.

All the templates can be in the form of a plate, a roller or roll orsheet, and can be in regard material or flexible. The template can befabricated by nanoprint. The template materials can be any materialsthat mechanical strong enough for templating and chemical inner enough.

Methods of Deposition.

The methods to shadow deposit materials can be any method, as long as itis more or less directional, and can evaporate the intended materials.The deposition methods include evaporation, sputtering and chemical ormolecular beams. The evaporation further includes the evaporation bychemical vapors, molecular beams, electron beam heating thermal heating,laser heating, and other heating methods. The sputtering includes thesputtering by ion, electron, plasmon, photon (i.e. laser), and otherenergetic particles.

Dip-Print.

The fabrication method of Dip-print is for patterning biodegradabledielectrics, since such materials cannot be thermally evaporated. Thefabrication method of Dip-print, as shown in FIG. 12, first puts a thinlayer of liquid polymer precursors with proper viscosity on a plate(termed “material transfer plate”); and then presses the template usedin Nano-PrinTED against the material transfer plate, picking up thepolymer precursors only at the top of the pillars of the template.Afterwards the polymer precursors will be cured to form a solid polymer.The dip-printed polymers will be used for the dielectrics between thestacked layers and for the dielectrics that glue different columns intoa single particle (Note different viscosity liquid will be useddepending upon the gap size between the columns).

The key advantages of this new fabrication technique are (a) form thecomplex structures that are needed for enhance plasmonic effects butcannot be formed in conventional fabrication methods, (b) have farbetter precision in controlling the NP structure dimensions (includingthe size and shape of each individual components, their spacing, andfinal particle), (c) a new way to solve the problem in patterningbiodegradable materials, and (d) scalable to large volume and low-cost(e.g. As having demonstrated, the fabrication rate is 2×10̂11 particlesper 4″ wafer per run, and it can be scaled up by over three orders ofmagnitude in throughput by roll-to-roll technology.) (ii) Fabrication:to advance new nanoparticle fabrication methods, nano-PrinTED andDip-print, and use them together with polymer chemistry to fabricate theS-particles with desired architectures, shapes, dimensions, andmaterials with high precision. One major goal is to achieve suchprecision fabrication for pillars of diameter of 6 nm or smaller, whichmeans to sub-6 nm size particles after biodegradation.

Surface Functioning for Sensing

The S-NP 100 becomes a nanosensor 200 after the surfacefunctionalization. Surface functioning of S-NP is to modify theproperties of the S-NP's surface to control the five key surfaceproperties: surface shape, chemical bonding, surface charge, hydrophobicand hydrophilic properties, and active targeting (FIG. 12).

Surface Shape.

The shape of a nanoparticle has effects of the NP's ability to enter andexit of a biological materials, such as cell and cell nuclei. Oneembodiment is to change S-NP shape after the fabrication as needed to adesired shape. The methods of changing the shape including coating apolymer or multilayer polymer, and biodegradable materials.

Chemical Bonding.

One of the most important surface chemistry modifications is to have thelinkers—the materials that link different kinds of biochemical reagent(e.g. targeting agent) onto nanoparticles. Bio-compatible blockco-polymer coatings, such as polyethylene glycol (PEG), will act as across-linker in this proposed research. We will investigate severalassembling methods, particularly two kinds. One kind is to function oneend of the polymer ligands with thiol-group to form strong bonding tothe nanoparticles' gold surface, and the other end of the linker withNHS-group to form strong covalent bond to the primary amine-site on theantibodies or proteins. We will also try to replace the thiol-end withsilane so that the cross-linker can be efficiently linked to dielectricsurface such as silica. The other method is using bio-affinity reactionssuch as avidin-biotin bonding. Other linkers are discussed in thesection of molecular adhesion layer.

Surface Charges and Wetting Properties.

The physicochemical characteristics of a polymeric nanoparticle such assurface charge and functional groups can affect its uptake by the cells.For phagocytic system, it is well accepted that positively chargednanoparticles have a higher rate of cell uptake compared to neutral ornegatively charged formulations (due to the negatively charged characterof the cell plasma membrane). The coating on nanoparticles for positivecharge is generally based on (or coated with) cationic polymers (e.g.the most widely used being the polysaccharide chitosan). In addition, weneed to give further consideration to the surface wetting properties ofthe coating material, because for biocompatible materials thatfacilitate biodegradation, it is preferred that the coating ishydrophilic and water soluble. We will choose PEG and PGA as thepositive and negative surface, respectively. Both materials are watersoluble, non-toxic, biodegradable and have long been used to passivatecolloid gold nanoparticle to facilitate both permeation and retention inbody [13, 14]. We will test the effect of charge on the S-nanoparticle'sbiodistribution and clearance on both cellular level and organism level.

Effect of Active Targeting.

We will use active targeting method, where we further enhance thedelivery specificity by conjugation of targeting ligands to the surfaceof nanoparticles. These ligands can include antibodies, engineeredantibody fragments, proteins, peptides, small molecules, and DNA or RNAaptamers. We will study the effect of active targeting on both cellularlevel and organism level.

Molecular Adhesion Layer

The capture agents for the target analytes are immobilized eitherdirectly on S-NPs 100 or through a molecular adhesion/spacer layer (MAL)160. As shown in FIG. 6, S-NP 100 comprises a molecular adhesion layer160 that covers at least a part of the metal surfaces of the underlyingS-NP. The molecular adhesion layer has two purposes. First, themolecular adhesion layer acts a spacer. For optimal fluorescence, thelight-emitting labels (e.g., fluorophores) cannot be too close to themetal surface because non-radiation processes would quench fluorescence.Nor can the light-emitting labels be too far from the metal surfacebecause it would reduce amplification. Ideally, the light-emittinglabels should be at an optimum distance from the metal surface. Second,the molecular adhesion layer provides a good adhesion to attach captureagent onto the S-NP. The good adhesion is achieved by having reactivegroups in the molecules of the molecular adhesion layer, which have ahigh affinity to the capture agent on one side and to the S-NPs on theother side.

The molecular adhesion layer (MAL) 160 can have many differentconfigurations, including (a) a self-assembled monolayer (SAM) ofcross-link molecules, (b) a multi-molecular layers thin film, (c) acombination of (a) and (b), and (d) a capture agent itself.

In the embodiment of MAL (a), where the molecular adhesion layer 160 isa self-assembled monolayer (SAM) of cross-link molecules or ligands,each molecule for the SAM comprises of three parts: (i) head group,which has a specific chemical affinity to the S-NP's surface, (ii)terminal group, which has a specific affinity to the capture agent, and(iii) molecule chain, which is a long series of molecules that link thehead group and terminal group, and its length (which determines theaverage spacing between the metal to the capture agent) can affect thelight amplification of the S-NP. Such a SAM is illustrated in FIG. 3.

In many embodiments, the head group attached to the metal surfacebelongs to the thiol group, e.t., —SH. Other alternatives for headgroups that attach to metal surface are, carboxylic acid (—COOH), amine(C═N), selenol (—SeH), or phosphane (—P). Other head groups, e.g. silane(—SiO), can be used if a monolayer is to be coated on dielectricmaterials or semiconductors, e.g., silicon.

In many embodiments, the terminal groups can comprise a variety ofcapture agent-reactive groups, including, but not limited to,N-hydroxysuccinimidyl ester, sulfo-N-hydroxysuccinimidyl ester, ahalo-substituted phenol ester, pentafluorophenol ester, anitro-substituted phenol ester, an anhydride, isocyanate,isothiocyanate, an imidoester, maleimide, iodoacetyl, hydrazide, analdehyde, or an epoxide. Other suitable groups are known in the art andmay be described in, e.g., Hermanson, “Bioconjugate Techniques” AcademicPress, 2nd Ed., 2008. The terminal groups can be chemically attached tothe molecule chain after they are assembled to the S-NP surface, orsynthesized together with the molecule chain before they are assembledon the surface.

Other terminal groups are Carboxyl —COOH groups (activated with EDC/NHSto form covalent binding with —NH2 on the ligand); Amine, —NH2, group(forming covalent binding with —COOH on the ligand via amide bondactivated by EDC/NHS); Epoxy, Reacted with the —NH2 (the ligand withoutthe need of a cross-linker); Aldehyde, (Reacted with the —NH2 on theligand without the need of a cross-linker); Thiol, —SH, (link to —NH2 onthe ligand through SMCC-like bioconjugation approach); and Glutathione,(GHS) (Ideal for capture of the GST-tagged proteins.

The molecular chain can be carbon chains, their lengths can be adjustedto change the distance between the light emitting label to the metal foroptimizing the optical signal. In one embodiment, as will be describedin greater detail in example section, the SAM layer isdithiobis(succinimidyl undecanoate), whose head group is —SH that bindsto gold surface through sulfer-gold bond, and terminal group isNHS-ester that bind to the primary amine sites of the capture agent, andthe molecule alkane chain with length of 1.7 nm.

In many embodiments, the molecule chains that link head groups andterminal groups are alkane chain, which is composed of only hydrogen andcarbon atoms, with all bonds are single bonds, and the carbon atoms arenot joined in cyclic structures but instead form a simple linear chain.Other alternatives for molecule chain can be ligands that are frompolymers such as poly(ethylene glycol) (PEG), Poly(lactic acid) (PLA),etc. The molecule chains are chemically non-reactive to neither themetal surface that the head groups attach to, nor the capture agent thatthe terminal groups attach to. The chain length, which determines thedistance of analyte to the S-NP's surface, can be optimized in order toachieve the maximum signal amplification. As will be described ingreater detail below, the molecule chains may have a thickness of, e.g.,0.5 nm to 50 nm.

The molecular adhesion layer used in the subject nanosensor may becomposed of a self-assembled monolayer (SAM) that is strongly attachedto the metal at one side (via, e.g., a sulfur atom) and that terminatesa capture-agent-reactive group, e.g., an amine-reactive group, athiol-reactive group, a hydroxyl-reactive group, an imidazolyl-reactivegroup and a guanidinyl-reactive group, at the other (exterior) side. Themonolayer may have a hydrophobic or hydrophilic surface. The mostcommonly used capture-agent reactive groups are NHS (which isamine-reactive) and maleimide (which is sulfhydryl-reactive), althoughmany others may be used.

In some embodiments, the molecular adhesion layer may be aself-assembled monolayer of an alkanethiol (see, e.g., Kato Journal ofPhysical Chemistry 2002 106: 9655-9658), poly(ethylene)glycol thiol(see, e.g., Shenoy et al Int. J. Nanomedicine. 2006 1: 51-57), anaromatic thiol or some other chain that terminates in the thiol.

Thiol groups may be used because (a) the thiol sulfur interacts withgold and other metals to form a bond that is both strong and stable bond(see, e.g., Nuzzo et al J. Am. Chem. Soc. 1987 109:2358-2368) and (b)van der Waals forces cause the alkane and other chains chains to stack,which causes a SAM to organize spontaneously (see, e.g., Love et al.Chem. Rev. 2006 105:1103-1169). Further, the terminal group is availablefor either direct attachment to the capture molecule or for furtherchemical modifications.

Alkanethiol may be used in some embodiments. It has been estimated thatthere are 4×10¹⁴ alkanethiol molecules/cm² in a packed monolayer ofalkanethiol (Nuzzo et al, J. Am. Chem. Soc. 1987 109:733-740), whichapproximately corresponds to an alkanethiol bond to every gold atom onthe underlying surface. Self-assembled monolayers composed ofalkanethiol can be generated by soaking the gold substrate in analkanethiol solution (see, e.g., Lee et al Anal. Chem. 2006 78:6504-6510). Gold is capable of reacting with both reduced alkanethiols(—SH groups) and alkyldisulfides(—S—S—) (see, e.g., Love et al Chem.Rev. 2005 105:1103-1169).

Once a self-assembled monolayer of poly(ethylene)glycol thiol oralkanethiol has been produced, a large number of strategies can beemployed to link a capture to the self-assembled monolayer. In oneembodiment, a capture agent such as streptavidin (SA) can be attached tothe SAM to immobilize biotinylated capture agents.

In one embodiment, streptavidin (SA) itself can be use as a functionalgroup (e.g. terminal group) the SAM to crosslink capture agent moleculesthat have high binding affinity to SA, such as biotinylated molecules,including peptides, oligonucleotides, proteins and sugars.

The functional group of avidin, streptavidin have a high affinity to thebiotin group to form avidin-biotin. Such high affinity makesavidin/streptavidin serve well as a functional group and the biotingroup as complementary functional group binding. Such functional groupcan be in binding the molecular adhesion layer to the S-NP, in bindingbetween molecular adhesion layer and the capature agent, and in bindinga light emitting lable to the secondary capture agent. In oneembodiment, a molecular adhesion layer containing thiol-reactive groupsmay be made by linking a gold surface to an amine-terminated SAM, andfurther modifying the amine groups usingsulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(Sulfo-SMCC) to yield a maleimide-activated surface. Maleimide-activatedsurfaces are reactive thiol groups and can be used to link to captureagents that contain thiol- (e.g., cysteine) groups.

In another embodiment, a molecular adhesion layer containing anamine-reactive group (N-hydroxl succinimide (NHS)) can be produced by,e.g., by soaking the gold substrate in a 1-10 mM solution ofsuccinimidyl alkanedisulfides such asdithiobis-sulfosuccinimidylpropionate (DSP) or dithiobis(succinimidylundecanoate) (see, e.g., Peelen et al J. Proteome Res. 2006 5:1580-1585and Storri et al Biosens. Bioelectron. 1998 13: 347-357).

In another embodiment, a molecular adhesion layer containing anamine-reactive group (NHS) may be produced using carboxyl-terminated SAMsuch as 12-carboxy-1-undecanethiol. In this case, the surface of the SAMmay be linked to the NHS in the presence of1-ethyl-3(3dimethylaminopropyl)carbodiimide HCl (EDC) to yield aninter-mediate which forms stable amide bonds with primary amines (see,e.g., Johnsson et al Anal. Biochem. 1001 198: 268-277).

In another embodiment, a molecular adhesion layer may contain Protein Awhich binds with high affinity to Fc region of IgGs, otherimmunoglobulin form, e.g., IgE.

In another embodiment, an imidazole group (which is also reactive withamines) may be added by reacting a carboxyl-terminated SAM with1,1′-carbonyldiimidazole (CDI).

In further embodiments, aldehyde-terminated alkanethiol monolayers canbe used to immobilize both proteins and amine-terminated DNAoligonucleotides, and his-tagged fusion proteins can be immobilized onnitrilotriacetic (NTA)-modified gold surfaces.

Thiol-reactive groups can link to synthetic DNA and RNAoligonucleotides, including aptamers, which can be readily synthesizedcommercially with a thiol terminus. Thiol-reactive groups can also linkto proteins that contain a cysteine groups, e.g., antibodies. Thiolatedmolecules can be attached to maleimide-modified surfaces (see, e.g.,Smith et al Langmuir 2002 19: 1486-1492). For in certain cases, one mayuse an amino acid spacer (e.g., Ser-Gly-Ser-Gly) inserted after aterminal Cys, which improves the amount of binding relative peptidesthat lacking spacers. For oligonucleotides, an alkane spacer can beused. Carbohydrates synthesized to contain with terminal thiols can bebeen tethered to gold in the same way.

Amine-reactive groups can form bonds with primary amines, such as thefree amine on lysine residues. In addition to proteins, amine-reactivesurfaces can be used to immobilize other biomolecules, includingpeptides containing lysine residues and oligonucleotides synthesizedwith an amine terminus.

In the embodiment of MAL (b), in which the molecular adhesion layer 160is a multi-molecular layer thin film, the molecules may be coated on theS-NP through physical adsorption or strong binding. In one example,protein A can be coated over the entire or partial areas of the surfaceof S-NP surface, in which case the protein A can be deposited throughphysical adsorption process and has a thickness of 4 nm to 5 nm. Inanother example, the layer may be a thin film of a polymer such aspolyethylene glycol (PEG), which has a functional head group on one end,e.g., thiol (—SH). The functioned PEG molecule layer forms a strong bondto S-NP's surface. The thickness of PEG molecule layer can be tuned bychanging the PEG polymer chain length. Another example is an amorphousSiO₂ thin film, which is attached to the surface of the S-NP usingphysical or chemical deposition methods, e.g., evaporation, sputtering,sol-gel method. The thickness of the SiO₂ thin film can be preciselycontrolled during the deposition.

In the embodiment of MAL (c), where the molecular adhesion layer 160 isa combination of a multi-molecular layer thin film and a SAM, the SAMlayer may be deposited first, followed by a multi-molecular layer.

In one example, the molecular adhesion layer may contain a monolayer ofstreptavidin first, followed by other layers of molecules that have highbinding affinity to streptavidin, such as biotin, biotinylatedmolecules, including peptides, oligonucleotides, proteins, and surgars.

In one example, the molecular adhesion layer, may contain a SAM layerdithiobis(succinimidyl undecanoate) (DSU) and a Protein A layer. The DSUSAM layer binds to S-NP's metal surface through sulfer-gold bond, andhas a terminal group of NHS-ester that binds to the primary amine siteson Protein A. In a particular case, capture antibodies bond to suchbilayer of protein A on top of DSU through their Fc region. The proteinA can ensure the orientation of antibodies for better captureefficiency.

In the embodiment of MAL (d), where the molecular adhesion layer 160 isa capture agent itself, the capture agent has a headgroup that have ahigh affinity to the metal or pillar sidewall of the subject S-NP. Oneof the common headgroup is thiol-reactive group. Thiol-reactive groupscan link to synthetic DNA and RNA oligonucleotides, including aptamers,which can be readily synthesized commercially with a thiol terminus.Thiol-reactive groups can also link to proteins that contain a cysteinegroups, e.g., antibodies. Another example where the MAL itself is usedas the capture agent is a layer of antibody fragments, e.g., half-IgG,Fab, F(ab′)2, Fc. The antibody fragments bond to metal surface directlythrough the thiol-endopeptidase located in the hinge region. Thisembodiment is illustrated in FIG. 8. In this embodiment, the nucleicacid comprises a headgroup that binds directly the S-NP. The remainderof the steps are performed as described in FIG. 7.

The thickness of molecular adhesion layer should be in the range of 0.5nm to 50 nm, e.g., 1 nm to 20 nm. The thickness of the molecularadhesion layer can be optimized to the particular application by, e.g.,increasing or decreasing the length of the linker (the alkane orpoly(ethylene glycol) chain) of the SAM used. Assuming each bond in thelinker is 0.1 nM to 0.15 nM, then an optimal SAM may contain a polymericlinker of 5 to 50 carbon atoms, e.g., 10 to 20 carbon atoms in certaincases.

A nanosensor may be made by attaching capture agents to the molecularadhesion layer via a reaction between the capture agent and acapture-agent reactive group on the surface of the molecular adhesionlayer.

Capture agents can be attached to the molecular adhesion layer via anyconvenient method such as those discussed above. In many cases, acapture agent may be attached to the molecular adhesion layer via ahigh-affinity strong interactions such as those between biotin andstreptavidin. Because streptavidin is a protein, streptavidin can belinked to the surface of the molecular adhesion layer using any of theamine-reactive methods described above. Biotinylated capture agents canbe immobilized by spotting them onto the streptavidin. In otherembodiments, a capture agent can be attached to the molecular adhesionlayer via a reaction that forms a strong bond, e.g., a reaction betweenan amine group in a lysine residue of a protein or an aminatedoligonucleotide with an NHS ester to produce an amide bond between thecapture agent and the molecular adhesion layer. In other embodiment, acapture agent can be strongly attached to the molecular adhesion layervia a reaction between a sulfhydryl group in a cysteine residue of aprotein or a sulfhydryl-oligonucleotide with a sulfhydryl-reactivemaleimide on the surface of the molecular adhesion layer. Protocols forlinking capture agents to various reactive groups are well known in theart.

In one embodiment, capture agent can be nucleic acid to captureproteins, or capture agent can be proteins that capture nucleic acid,e.g., DNA, RNA. Nucleic acid can bind to proteins throughsequence-specific (tight) or non-sequence specific (loose) bond.

In certain instances, a subject S-NP may be fabricated using the method:(a) patterning at least one pillar on a top surface of a substrate; (b)depositing a metallic material layer of the top surface; (c) allowingthe metallic material deposited on the pillar tops to form a disc, themetallic material deposited on the pillar feet to form a metallic backplane, and the metallic material deposited on the sidewall to form atleast one metallic dot structure; and, as described above, (d)depositing a molecular adhesion layer on top of the deposited metallicmaterial, wherein the molecular adhesion layer covers at least a part ofthe metallic dot structure, the metal disc, and/or the metallic backplane, and wherein the exterior surface of the molecular adhesion layercomprises a capture agent-reactive group.

Furthermore, the patterning in (a) include a direct imprinting(embossing) of a material, which can be dielectric or semiconductor inelectric property, and can be polymers or polymers formed by curing ofmonomers or oligomers, or amorphous inorganic materials. The materialcan be a thin film with a thickness from 10 nanometer to 10 millimeter,or multilayer materials with a substrate. The imprinting (i.e.embossing) means to have mold with a structure on its surface, and pressthe mold into the material to be imprinted to for an inverse of thestructure in the material. The substrates or the top imprinted layerscan be a plastic (i.e. polymers), e.g. polystyring (PS), Poly(methylmethacrylate) (PMMA), Polyethylene terephthalate (PET), other acrylics,and alike. The imprinting may be done by roll to roll technology using aroller imprinter. Such process has a great economic advantage and hencelowering the cost.

Sensing Systems

Also provided is a system comprising a subject nanosensor, a holder forthe nanosensor, an excitation source that induces a light signal from alabel (i.e. light emitting label); and a reader (e.g., a photodetector,a CCD camera, a CMOS camera, a spectrometer or an imaging device capableof producing a two dimensional spectral map of a surface of thenanosensor) adapted to read the light signal. As would be apparent, thesystem may also has electronics, computer system, software, and otherhardware that amplify, filter, regulate, control and store theelectrical signals from the reader, and control the reader and sampleholder positions. The sample holder position can be move in one or allthree orthogonal directions to allow the reader to scan the light signalfrom different locations of the sample.

The excitation source may be (a) a light source, e.g., a laser of awavelength suitable for exciting a particular fluorophore, and a lamp ora light emitting diode with a light filter for wavelength selection; or(b) a power source for providing an electrical current to excite lightout of the nanosensor (which may be employed when anelectrochemiluminescent label is used).

In particular cases, laser-line pass filter filters out light whosewavelength is different from the laser, and the long wavelength passfilter will only allow the light emanate from the optically detectablelabel to pass through. Since different fluorescence labels absorb lightin different spectral range, the fluorescence label should be chosen tomatch its peak absorption wavelength to the laser excitation wavelengthin order to achieve optimum quantum efficiency. In many embodiments, thelight signal emanating from the fluorescence label on the nanosensorsare at a wavelength of at least 20 nm higher than the laser wavelength.Thus the nanosensor's plasmonic resonance should be tuned to cover thefluorescence label's abosprtion peak, emission peak and laser excitationwavelength. In some embodiments, the excitation and fluorescencewavelength range can be from 100 nm to 20,000 nm. The preferred range isfrom 300 nm to 1200 nm. The 600-850 nm range is preferable due to lowbackground noise.

It is apparent there are other ways to achieve the functions of lightexcitation and reading.

As would be apparent from the above, certain nanosensors may beimplemented in a multi-well format. In these embodiments, the stage canmove moved so that reader can read a light signal from each of the wellsof the multi-well plate, independently.

Applications in Chemical and Biological Sensing and Assay Methods

The functionalized S-particles can be used as biological and chemicalsensing, including detection of biological and chemical markers, suchproteins, DNAs, RNAs, and other organic and inorganic molecules, insingle cells, tissue, and in-vivo for human and animals, and diagnosis.

Here diagnosis means to assess the condition of certain disease orcondition by quantitative detection of certain biomarkers orbiomolecules. Such diagnosis by S-NPs include detection of proteins,nucleic acids, micro-organisms (virus, bacteria, etc.) and mallmolecules (hormones, etc.).

The methods of diagnosis by S-NPs include In vitro detection,fluorescence based detection, homogeneous fluorescence immunoassay(Alpha-LISA), flow-cytometery based detection, colorimetric detection,bio-bar-code Assay, SERS-based detection, SERS label homogeneousimmunoassay, multiplex SERS label, in vivo detection, fluorescenceImaging (e.g. tumor cells), optical tomography and MRI.

The subject nanosensor may be used to detect analytes in a sample. Thismethod may comprise: (a) contacting a sample comprising an analyte witha nanosensor under conditions suitable for specific binding of ananalyte in the sample with the capture agent; and (b) reading anoptically detectable signal from the nanosensor, wherein the opticallydetectable signal indicates that the analyte is bound to the captureagent. In the above step (a), before the bonding to the capture agent,the analyte may be labeled with a light-emitting label or not labeled(also referred as labeled directly or indirectly). In embodiments inwhich an analyte is no labeled with a light-emitting label before thebonding, the analyte, after the bonding to the capture agent, may bebound to a second capture agent (i.e. detection agent) (e.g., asecondary antibody or another nucleic acid) that is itself opticallylabeled, labeled secondary capture agent or labeled detection agent,(such process is also referred as indirectly labeling of an analyte). Ina sensing using indirectly labeling, the labeled secondary captureagents unbounded to analytes are removed before the above reading step(b). In a sensing using directly labeling, the optical labels unboundedto analytes are removed before the above reading step (b).

In reading the light emitting labels on the assay, an excitation (photo,electro, chemical or combination of them) are applied to light emittinglabel, and the properties of light including intensity, wavelength, andlocation are detected.

In certain embodiments, the method comprises attaching a capture agentto the molecular adhesion layer of a subject S-NP to produce ananosensor, wherein the attaching is done via a chemical reaction of thecapture agent with the capture agent-reactive group in the molecules onthe molecular adhesion layer, as described above. Next, the methodcomprises contacting a sample containing a target-analyte with thenanosensor and the contacting is done under conditions suitable forspecific binding and the target-analyte specifically binds to thecapture agent. After this step, the method comprises removing anytarget-analytes that are not bound to the capture agent (e.g., bywashing the surface of the nanosensor in binding buffer); Then detectionagent conjugated with optical detectable label is added to detect thetarget-analyte. After removing the detection agent that are not bound tothe target-analyte, The S-NP can then be used, with a reading system, toread a light signal (e.g., light at a wavelength that is in the range of300 nm to 1200 nm) from detection agent that remain bound to thenanosensor. As would be apparent, the method further comprises labelingthe target analytes with a light-emitting label. This can be done eitherprior to or after the contacting step, i.e., after the analytes arebound to the capture agent. In certain embodiments, analytes are labeledbefore they are contacted with the nanosensor. In other embodiment, theanalytes are labeled after they are bound to the capture agents of thenanosensor. Further, as mentioned above, the analyte may be labeleddirectly (in which case the analyte may be strongly linked to alight-emitting label at the beginning of the method), or labeledindirectly (i.e., by binding the target analytes to a second captureagent, e.g., a secondary antibody that is labeled or a labeled nucleicacid, that specifically binds to the target analyte and that is linkedto a light-emitting label). In some embodiments, the method may compriseblocking the nanosensor prior to the contacting step (b), therebypreventing non-specific binding of the capture agents to non-targetanalytes.

The suitable conditions for the specific binding and the target-analytespecifically binds to the capture agent, include proper temperature,time, solution pH level, ambient light level, humidity, chemical reagentconcentration, antigen-antibody ratio, etc.

In certain embodiments, a nucleic acid capture agent can be used tocapture a protein analyte (e.g., a DNA or RNA binding protein). Inalternative embodiments, the protein capture agent (e.g., a DNA or RNAbinding protein) can be used to capture a nucleic acid analyte.

The sample may be a liquid sample and, in certain embodiments, thesample may be a clinical sample derived from cells, tissues, or bodilyfluids. Bodily fluids of interest include but are not limited to,amniotic fluid, aqueous humour, vitreous humour, blood (e.g., wholeblood, fractionated blood, plasma, serum, etc.), breast milk,cerebrospinal fluid (CSF), cerumen (earwax), chyle, chime, endolymph,perilymph, feces, gastric acid, gastric juice, lymph, mucus (includingnasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleuralfluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, sweat,synovial fluid, tears, vomit, urine and exhaled condensate.

Some of the steps of an assay are shown in FIGS. 7 and 8. Generalmethods for methods for molecular interactions between capture agentsand their binding partners (including analytes) are well known in theart (see, e.g., Harlow et al., Antibodies: A Laboratory Manual, FirstEdition (1988) Cold spring Harbor, N.Y.; Ausubel, et al, Short Protocolsin Molecular Biology, 3rd ed., Wiley & Sons, 1995). The methods shown inFIGS. 4 and 5 are exemplary; the methods described in those figures arenot the only ways of performing an assay.

Some of the steps of an exemplary antibody binding assay are shown inFIG. 5. In this assay, S-NP 100 is linked to an antibody in accordancewith the methods described above to produce a nanosensor 200 thatcomprises antibodies 202 that are linked to the molecular adhesion layerof the S-NP. After nanosensor 200 has been produced, the nanosensor iscontacted with a sample containing a target analyte (e.g., a targetprotein) under conditions suitable for specific binding. The antibodies202 specifically bind to target analyte 204 in the sample. After unboundanalytes have been washed from the nanosensor, the nanosensor iscontacted with a secondary antibody 206 that is labeled with alight-emitting label 208 under conditions suitable for specific binding.After unbound secondary antibodies have been removed from thenanosensor, the nanosensor may be read to identify and/or quantify theamount of analyte 204 in the initial sample.

Some of the steps of an exemplary nucleic acid binding assay are shownin FIGS. 6 and 7. In this assay, S-NP 100 is linked to a nucleic acid,e.g., an oligonucleotide in accordance with the methods described aboveto produce a nanosensor 300 that comprises nucleic acid molecules 302that are linked to the molecular adhesion layer. After nanosensor 300has been produced, the nanosensor is contacted with a sample containingtarget nucleic acid 304 under conditions suitable for specifichybridization of target nucleic acid 304 to the nucleic acid captureagents 302. Nucleic acid capture agents 304 specifically binds to targetnucleic acid 304 in the sample. After unbound nucleic acids have beenwashed from the nanosensor, the nanosensor is contacted with a secondarynucleic acid 306 that is labeled with a light-emitting label 308 underconditions for specific hybridization. After unbound secondary nucleicacids have been removed from the nanosensor, the nanosensor may be readto identify and/or quantify the amount of nucleic acid 304 in theinitial sample.

One example of an enhanced DNA hybridization assay that can be performedusing a subject device is a sandwich hybridization assay. The captureDNA is a single strand DNA functioned with thiol at its 3′-end Thedetection DNA is a single strand DNA functioned with a fluorescencelabel e.g., IRDye800CW at its 3′-end. Both the capture and detection DNAhas a length of 20 bp. They are synthesized with different sequences toform complementary binding to a targeted DNA at different region. Firstthe capture DNA is immobilized on the S-NP's metal surface throughsulfur-gold reaction. Then targeted DNA is added to the S-NP to becaptured by the capture DNA. Finally the fluorescence labeled detectionDNA is added to the S-NP to detect the immobilized targeted DNA. Afterwashing off the unbound detection DNA, the fluorescence signal emanatefrom the S-NPs' surface is measured for the detection and quantificationof targeted DNA molecules.

In the embodiments shown in FIGS. 5 and 6, bound analyte can be detectedusing a secondary capture agent (i.e. the “detection agent”) may beconjugated to a fluorophore or an enzyme that catalyzes the synthesis ofa chromogenic compound that can be detected visually or using an imagingsystem. In one embodiment, horseradish peroxidase (HRP) may be used,which can convert chromogenic substrates (e.g., TMB, DAB, or ABTS) intocolored products, or, alternatively, produce a luminescent product whenchemiluminescent substrates are used. In particular embodiments, thelight signal produced by the label has a wavelength that is in the rangeof 300 nm to 900 nm). In certain embodiments, the label may beelectrochemiluminescent and, as such, a light signal can be produced bysupplying a current to the sensor.

In some embodiments, the secondary capture agent (i.e. the detectionagent), e.g., the secondary antibody or secondary nucleic acid, may belinked to a fluorophore, e.g., xanthene dyes, e.g. fluorescein andrhodamine dyes, such as fluorescein isothiocyanate (FITC),6-carboxyfluorescein (commonly known by the abbreviations FAM and F),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE or J),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA or T),6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G⁵ or G⁵),6-carboxyrhodamine-6G (R6G⁶ or G⁶), and rhodamine 110; cyanine dyes,e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimidedyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidiumdyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes;polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc; BODIPY dyesand quinoline dyes. Specific fluorophores of interest that are commonlyused in subject applications include: Pyrene, Coumarin,Diethylaminocoumarin, FAM, Fluorescein Chlorotriazinyl, Fluorescein,R110, Eosin, JOE, R6G, Tetramethylrhodamine, TAMRA, Lissamine, ROX,Napthofluorescein, Texas Red, Napthofluorescein, Cy3, and Cy5, IRDye800,IRDye800CW, Alexa 790, Dylight 800, etc.

The primary and secondary capture agents should bind to the targetanalyte with highly-specific affinity. However, the primary andsecondary capture agents cannot be the molecule because they need tobind to different sites in the antigen. One example is the anti-humanbeta amyloid capture antibody 6E10 and detection G210, in which case6E10 binds only to the 10^(th) amine site on human beta amyloids peptidewhile G210 binds only to the 40^(th) amine site. Capture agent andsecondary capture agent do not react to each other. Another example usesrabbit anti-human IgG as capture antibody and donkey anti-human IgG asdetection antibody. Since the capture and detection agents are derivedfrom different host species, they do not react with each other.

Methods for labeling proteins, e.g., secondary antibodies, and nucleicacids with fluorophores are well known in the art. Chemiluminescentlabels include acridinium esters and sulfonamides, luminol andisoluminol; electrochemiluminescent labels include ruthenium (II)chelates, and others are known.

Applications

The subject methods and compositions find use in a variety applications,where such applications are generally analyte detection applications inwhich the presence of a particular analyte in a given sample is detectedat least qualitatively, if not quantitatively. Protocols for carryingout analyte detection assays are well known to those of skill in the artand need not be described in great detail here. Generally, the samplesuspected of comprising an analyte of interest is contacted with thesurface of a subject nanosensor under conditions sufficient for theanalyte to bind to its respective capture agent that is tethered to thesensor. The capture agent has highly specific affinity for the targetedmolecules of interest. This affinity can be antigen-antibody reactionwhere antibodies bind to specific epitope on the antigen, or a DNA/RNAor DNA/RNA hybridization reaction that is sequence-specific between twoor more complementary strands of nucleic acids. Thus, if the analyte ofinterest is present in the sample, it likely binds to the sensor at thesite of the capture agent and a complex is formed on the sensor surface.Namely, the captured analytes are immobilized at the sensor surface.After removing the unbounded analytes, the presence of this bindingcomplex on the surface of the sensor (i.e. the immobilized analytes ofinterest) is then detected, e.g., using a labeled secondary captureagent.

Specific analyte detection applications of interest includehybridization assays in which the nucleic acid capture agents areemployed and protein binding assays in which polypeptides, e.g.,antibodies, are employed. In these assays, a sample is first preparedand following sample preparation, the sample is contacted with a subjectnanosensor under specific binding conditions, whereby complexes areformed between target nucleic acids or polypeptides (or other molecules)that are complementary to capture agents attached to the sensor surface.

In one embodiment, the capture oligonucleotide is synthesized singlestrand DNA of 20-100 bases length, that is thiolated at one end. Thesemolecules are are immobilized on the S-NPs' surface to capture thetargeted single-strand DNA (which may be at least 50 bp length) that hasa sequence that is complementary to the immobilized capture DNA. Afterthe hybridization reaction, a detection single strand DNA (which can beof 20-100 bp in length) whose sequence are complementary to the targetedDNA's unoccupied nucleic acid is added to hybridize with the target. Thedetection DNA has its one end conjugated to a fluorescence label, whoseemission wavelength are within the plasmonic resonance of the S-NP.Therefore by detecting the fluorescence emission emanate from the S-NPs'surface, the targeted single strand DNA can be accurately detected andquantified. The length for capture and detection DNA determine themelting temperature (nucleotide strands will separate above meltingtemperature), the extent of misparing (the longer the strand, the lowerthe misparing). One of the concerns of choosing the length forcomplementary binding depends on the needs to minimize misparing whilekeeping the melting temperature as high as possible. In addition, thetotal length of the hybridization length is determined in order toachieve optimum signal amplification.

A subject sensor may be employed in a method of diagnosing a disease orcondition, comprising: (a) obtaining a liquid sample from a patientsuspected of having the disease or condition, (b) contacting the samplewith a subject nanosensor, wherein the capture agent of the nanosensorspecifically binds to a biomarker for the disease and wherein thecontacting is done under conditions suitable for specific binding of thebiomarker with the capture agent; (c) removing any biomarker that is notbound to the capture agent; and (d) reading a light signal frombiomarker that remain bound to the nanosensor, wherein a light signalindicates that the patient has the disease or condition, wherein themethod further comprises labeling the biomarker with a light-emittinglabel, either prior to or after it is bound to the capture agent. Aswill be described in greater detail below, the patient may suspected ofhaving cancer and the antibody binds to a cancer biomarker. In otherembodiments, the patient is suspected of having a neurological disorderand the antibody binds to a biomarker for the neurological disorder.

The applications of the subject sensor include, but not limited to, (a)the detection, purification and quantification of chemical compounds orbiomolecules that correlates with the stage of certain diseases, e.g.,infectious and parasitic disease, injuries, cardiovascular disease,cancer, mental disorders, neuropsychiatric disorders and organicdiseases, e.g., pulmonary diseases, renal diseases, (b) the detection,purification and quantification of microorganism, e.g., virus, fungusand bacteria from environment, e.g., water, soil, or biological samples,e.g., tissues, bodily fluids, (c) the detection, quantification ofchemical compounds or biological samples that pose hazard to food safetyor national security, e.g. toxic waste, anthrax, (d) quantification ofvital parameters in medical or physiological monitor, e.g., glucose,blood oxygen level, total blood count, (e) the detection andquantification of specific DNA or RNA from biosamples, e.g., cells,viruses, bodily fluids, (f) the sequencing and comparing of geneticsequences in DNA in the chromosomes and mitochondria for genome analysisor (g) to detect reaction products, e.g., during synthesis orpurification of pharmaceuticals.

The detection can be carried out in various sample matrix, such ascells, tissues, bodily fluids, and stool. Bodily fluids of interestinclude but are not limited to, amniotic fluid, aqueous humour, vitreoushumour, blood (e.g., whole blood, fractionated blood, plasma, serum,etc.), breast milk, cerebrospinal fluid (CSF), cerumen (earwax), chyle,chime, endolymph, perilymph, feces, gastric acid, gastric juice, lymph,mucus (including nasal drainage and phlegm), pericardial fluid,peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil),semen, sputum, sweat, synovial fluid, tears, vomit, urine and exhaledcondensate.

In some embodiments, a subject biosensor can be used diagnose a pathogeninfection by detecting a target nucleic acid from a pathogen in asample. The target nucleic acid may be, for example, from a virus thatis selected from the group comprising human immunodeficiency virus 1 and2 (HIV-1 and HIV-2), human T-cell leukaemia virus and 2 (HTLV-1 andHTLV-2), respiratory syncytial virus (RSV), adenovirus, hepatitis Bvirus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), humanpapillomavirus (HPV), varicella zoster virus (VZV), cytomegalovirus(CMV), herpes-simplex virus 1 and 2 (HSV-1 and HSV-2), human herpesvirus8 (HHV-8, also known as Kaposi sarcoma herpesvirus) and flaviviruses,including yellow fever virus, dengue virus, Japanese encephalitis virusand West Nile virus. The present invention is not, however, limited tothe detection of DNA sequences from the aforementioned viruses, but canbe applied without any problem to other pathogens important inveterinary and/or human medicine.

Human papillomaviruses (HPV) are further subdivided on the basis oftheir DNA sequence homology into more than 70 different types. Thesetypes cause different diseases. HPV types 1, 2, 3, 4, 7, 10 and 26-29cause benign warts. HPV types 5, 8, 9, 12, 14, 15, 17 and 19-25 and46-50 cause lesions in patients with a weakened immune system. Types 6,11, 34, 39, 41-44 and 51-55 cause benign acuminate warts on the mucosaeof the genital region and of the respiratory tract. HPV types 16 and 18are of special medical interest, as they cause epithelial dysplasias ofthe genital mucosa and are associated with a high proportion of theinvasive carcinomas of the cervix, vagina, vulva and anal canal.Integration of the DNA of the human papillomavirus is considered to bedecisive in the carcinogenesis of cervical cancer. Humanpapillomaviruses can be detected for example from the DNA sequence oftheir capsid proteins L1 and L2. Accordingly, the method of the presentinvention is especially suitable for the detection of DNA sequences ofHPV types 16 and/or 18 in tissue samples, for assessing the risk ofdevelopment of carcinoma.

In some cases, the nanosensor may be employed to detect a biomarker thatis present at a low concentration. For example, the nanosensor may beused to detect cancer antigens in a readily accessible bodily fluids(e.g., blood, saliva, urine, tears, etc.), to detect biomarkers fortissue-specific diseases in a readily accessible bodily fluid (e.g., abiomarkers for a neurological disorder (e.g., Alzheimer's antigens)), todetect infections (particularly detection of low titer latent viruses,e.g., HIV), to detect fetal antigens in maternal blood, and fordetection of exogenous compounds (e.g., drugs or pollutants) in asubject's bloodstream, for example.

The following table provides a list of protein biomarkers that can bedetected using the subject nanosensor (when used in conjunction with anappropriate monoclonal antibody), and their associated diseases. Onepotential source of the biomarker (e.g., “CSF”; cerebrospinal fluid) isalso indicated in the table. In many cases, the subject biosensor candetect those biomarkers in a different bodily fluid to that indicated.For example, biomarkers that are found in CSF can be identified inurine, blood or saliva, for example.

Marker disease Aβ42, amyloid beta-protein (CSF) Alzheimer's disease.fetuin-A (CSF) multiple sclerosis. tau (CSF) niemann-pick type C.secretogranin II (CSF) bipolar disorder. prion protein (CSF) Alzheimerdisease, prion disease Cytokines (CSF) HIV-associated neurocognitivedisorders Alpha-synuclein (CSF) parkinsonian disorders(neuordegenerative disorders) tau protein (CSF) parkinsonian disordersneurofilament light chain (CSF) axonal degeneration parkin (CSF)neuordegenerative disorders PTEN induced putative kinase 1 (CSF)neuordegenerative disorders DJ-1 (CSF) neuordegenerative disordersleucine-rich repeat kinase 2 (CSF) neuordegenerative disorders mutatedATP13A2 (CSF) Kufor-Rakeb disease Apo H (CSF) parkinson disease (PD)ceruloplasmin (CSF) PD Peroxisome proliferator-activated receptor PDgamma coactivator-1 alpha (PGC-1α)(CSF) transthyretin (CSF) CSFrhinorrhea (nasal surgery samples) Vitamin D-binding Protein (CSF)Multiple Sclerosis Progression proapoptotic kinase R (PKR) and its ADphosphorylated PKR (pPKR) (CSF) CXCL13 (CSF) multiple sclerosisIL-12p40, CXCL13 and IL-8 (CSF) intrathecal inflammation Dkk-3 (semen)prostate cancer p14 endocan fragment (blood) Sepsis: Endocan,specifically secreted by activated-pulmonary vascular endothelial cells,is thought to play a key role in the control of the lung inflammatoryreaction. Serum (blood) neuromyelitis optica ACE2 (blood) cardiovasculardisease autoantibody to CD25 (blood) early diagnosis of esophagealsquamous cell carcinoma hTERT (blood) lung cancer CAI25 (MUC 16) (blood)lung cancer VEGF (blood) lung cancer sIL-2 (blood) lung cancerOsteopontin (blood) lung cancer Human epididymis protein 4 (HE4) (blood)ovarian cancer Alpha-Fetal Protein (blood) pregnancy Albumin (urine)diabetics albumin (urine) uria albuminuria microalbuminuria kidney leaksAFP (urine) mirror fetal AFP levels neutrophil gelatinase-associatedlipocalin (NGAL) Acute kidney injury (urine) interleukin 18 (IL-18)(urine) Acute kidney injury Kidney Injury Molecule -1 (KIM-1) (urine)Acute kidney injury Liver Fatty Acid Binding Protein (L-FABP) (urine)Acute kidney injury LMP1 (saliva) Epstein-Barr virus oncoprotein(nasopharyngeal carcinomas) BARF1 (saliva) Epstein-Barr virusoncoprotein (nasopharyngeal carcinomas) IL-8 (saliva) oral cancerbiomarker carcinoembryonic antigen (CEA) (saliva) oral or salivarymalignant tumors BRAF, CCNI, EGRF, FGF19, FRS2, GREB1, and Lung cancerLZTS1 (saliva) alpha-amylase (saliva) cardiovascular diseasecarcinoembryonic antigen (saliva) Malignant tumors of the oral cavity CA125 (saliva) Ovarian cancer IL8 (saliva) spinalcellular carcinoma.thioredoxin (saliva) spinalcellular carcinoma. beta-2 microglobulinlevels - monitor activity of HIV the virus (saliva) tumor necrosisfactor-alpha receptors - monitor HIV activity of the virus (saliva)CA15-3 (saliva) breast cancer

As noted above, a subject nanosensor can be used to detect nucleic acidin a sample. A subject nanosensor may be employed in a variety of drugdiscovery and research applications in addition to the diagnosticapplications described above. For example, a subject nanosensor may beemployed in a variety of applications that include, but are not limitedto, diagnosis or monitoring of a disease or condition (where thepresence of an nucleic acid provides a biomarker for the disease orcondition), discovery of drug targets (where, e.g., an nucleic acid isdifferentially expressed in a disease or condition and may be targetedfor drug therapy), drug screening (where the effects of a drug aremonitored by assessing the level of an nucleic acid), determining drugsusceptibility (where drug susceptibility is associated with aparticular profile of nucleic acids) and basic research (where is itdesirable to identify the presence a nucleic acid in a sample, or, incertain embodiments, the relative levels of a particular nucleic acidsin two or more samples).

In certain embodiments, relative levels of nucleic acids in two or moredifferent nucleic acid samples may be obtained using the above methods,and compared. In these embodiments, the results obtained from theabove-described methods are usually normalized to the total amount ofnucleic acids in the sample (e.g., constitutive RNAs), and compared.This may be done by comparing ratios, or by any other means. Inparticular embodiments, the nucleic acid profiles of two or moredifferent samples may be compared to identify nucleic acids that areassociated with a particular disease or condition.

In some examples, the different samples may consist of an “experimental”sample, i.e., a sample of interest, and a “control” sample to which theexperimental sample may be compared. In many embodiments, the differentsamples are pairs of cell types or fractions thereof, one cell typebeing a cell type of interest, e.g., an abnormal cell, and the other acontrol, e.g., normal, cell. If two fractions of cells are compared, thefractions are usually the same fraction from each of the two cells. Incertain embodiments, however, two fractions of the same cell may becompared. Exemplary cell type pairs include, for example, cells isolatedfrom a tissue biopsy (e.g., from a tissue having a disease such ascolon, breast, prostate, lung, skin cancer, or infected with a pathogenetc.) and normal cells from the same tissue, usually from the samepatient; cells grown in tissue culture that are immortal (e.g., cellswith a proliferative mutation or an immortalizing transgene), infectedwith a pathogen, or treated (e.g., with environmental or chemical agentssuch as peptides, hormones, altered temperature, growth condition,physical stress, cellular transformation, etc.), and a normal cell(e.g., a cell that is otherwise identical to the experimental cellexcept that it is not immortal, infected, or treated, etc.); a cellisolated from a mammal with a cancer, a disease, a geriatric mammal, ora mammal exposed to a condition, and a cell from a mammal of the samespecies, preferably from the same family, that is healthy or young; anddifferentiated cells and non-differentiated cells from the same mammal(e.g., one cell being the progenitor of the other in a mammal, forexample). In one embodiment, cells of different types, e.g., neuronaland non-neuronal cells, or cells of different status (e.g., before andafter a stimulus on the cells) may be employed. In another embodiment ofthe invention, the experimental material is cells susceptible toinfection by a pathogen such as a virus, e.g., human immunodeficiencyvirus (HIV), etc., and the control material is cells resistant toinfection by the pathogen. In another embodiment of the invention, thesample pair is represented by undifferentiated cells, e.g., stem cells,and differentiated cells.

Although the foregoing embodiments have been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the above teachings that certain changes andmodifications can be made thereto without departing from the spirit orscope of the appended claims.

EXAMPLES

Various S-NP particles have been fabricated by Nano-PrinTED andDip-print and tested for enhancements of both SERS and fluorescence(tested with light wavelength of ˜800 nm). Exemplary results are givenbelow.

For the S-NP structure with light resonance absorption around 800 nmwavelength, the disks have a round shape of diameter of 70 nm, the topmetallic (gold) disk thickness is 15 nm, the spacer (silicon dioxide)thickness is 20 nm, and the bottom metallic (gold) disk thickness is 15nm, the self-assembled gold dots diameter is around 10 nm, and theadhesion layers between the disks are titanium of a thickness of 0.5 nm.

In fabrication, first, the dense nanostructured protrusions templatewith was patterned on 4 inch substrate by nanoimprint lithography andreactive ion etching (RIE). The pillar height or etching depth wasprecisely controlled by RIE (FIG. 15 a). The mold can be a daughter moldduplicated by nanoimprint from a master mold fabricated withinterference lithography, e-beam lithography, sphere lithography andothers. Second, the multiple depositions: a release layer, metal layers(e.g. gold), adhesion layers (e.g. titanium), dielectric layers (e.g.silicon dioxide) were deposited in a sequence onto the protrusionstemplate in a normal direction to the surface by evaporation as shown inFIG. 15 b. Guided by the protrusions, the materials deposited on the topformed DS-NP arrays, and at the pillar foot, a multi-layer nano-holebackplane was also formed, the two are not connected. Third,transfer-print S-NPs to another substrate (FIG. 15 c), presents theschematic of the S-NPs array that has been transferred onto anothersubstrate. Before transferring, a thin layer of buffer layer around50-100 nm was spinned onto the substrate served as an adhesion layer.The transferring process was taken under low pressure (e.g. 50 PSI) androom temperature, thus not damaged the substrate nor D2-Particle arrays.Then the template peeled off the S-NPs from the templates. And fourth,solvent was used to dissolve the buffer layer and released the S-NPsinto the solutions to make S-NPs (FIG. 15 d).

FIG. 13 shows the scanning electron microscopy (SEMs) of (a)double-metal-disk and single dielectric (D-particle); (b) triple-metal(or magnetic) dielectric-nanoparticle (TS-NP); (c) D-particle after theself-perfection by liquefaction (SPEL) to change the shape of 2 metaldisks; (d) D-particles array on the substrate after the templatelift-off.

FIG. 14 shows the scanning electron microscopy (SEMs) of (a) D-particlesarray on the substrate after the transfer printing. (b) D-particlesexfoliated into solution.

FIG. 15 shows the Nano-PrinTED (nanoprint by templated exfoliateabledeposition) fabrication of S-NP at each step. Top row: Schematic. Andbottom row: scanning electron microscope (SEM) of experimental results.(a) Pillar template fabricated by lithography (e.g. NIL); (b) Multipledeposition and self-assembly to form D2-particles; (c) transfer-printDPs to another substrate; (d) put in solution. (e-h), SEM images.

As shown in FIG. 16, Nano-PrinTED and Dip-print have far betterprecision in controlling the NP structure dimensions (including the sizeand shape of each individual components, their spacing, and finalparticle). (a) SEM picture of D2-P before release and (b) Measured sizedistribution. Measured size variation of D2-particle fabricated byNano-PrinTED (<5%) is 3 fold less than AuNP manufactured by chemicalsynthesis (>15%).

FIG. 17 shows the measurements of extinction spectrum of D2-particleswith SiO₂ layer thickness from 5 nm to 30 nm and constant Au layerthicknesses of 20 nm. Plasmonic resonant peak wavelengths redshift withincreasing SiO₂ layer thickness.

FIG. 18 shows the simulation of the size of nanoparticles with differentarchitectures required for the same resonant wavelength at 800 nm. Itclearly shows that S-NP has much smaller particle sized thanconventional metallic sphere and disks for a given resonant wavelength.

As shown in FIG. 19, (a) Measured Surface Enhanced Raman Spectroscopy(SERS) signal of BPE, and (b) fluorescence signal of IR-800 dye withsingle D2-particle and gold nanoparticle. A single D2-particle has aSERS/Fluorescence enhancement over 100/30 fold higher than a single goldnanoparticle of similar diameter. The sophisticated architectures ofPDS-NPs allow simultaneously improving of all three key factors forplasmonic enhancement and hence a large final enhancement. In PDS-NPs,the metallic disks (25-60 nm diameter) create antenna for goodabsorption of excitation light and radiation of the generated opticalsignal, while the smaller gaps (between the disks or additionalnanodots) and sharp edges offer large local field enhancements.

What is claimed is:
 1. A nanoparticle that enhances the interaction ofthe nanoparticle and/or a molecule/material deposited on the surface ofthe nanoparticle with light, comprising a pair of stacked metallic disksseparated by a non-metallic spacer, wherein: (a) the dimensions of thedisks and spacer are smaller than the wavelength of the light; and (b)the nanoparticle enhance the light interaction at least three timesgreater than that an individual metallic disk.
 2. The nanoparticle ofany prior claim, wherein the light interaction includes lightabsorption, light scattering, light reflection, and light radiation. 3.The nanoparticle of any prior claim, wherein the light interactionincludes Raman scattering, color production, and luminescence thatincludes fluorescence, electroluminescence, chemiluminescence, andelectrochemiluminescence.
 4. The nanoparticle of any prior claim,wherein the light interaction comprises interactions of light with amaterials or molecule that is deposited on the nanoparticle.
 5. Thenanoparticle of claim 4, wherein the molecules are analytes that havebeen captured on the surface of the nanoparticle.
 6. The nanoparticle ofclaim 4 or 5, wherein the analytes are selected from proteins, peptides,DNA, RNA, nucleic acid, small molecules, cells, and nanoparticles withdifferent shape.
 7. The nanoparticle of any prior claim, wherein thenanoparticle further comprises two masking layers covering the exteriorsurfaces of the metallic disks but a portion of the edges of the disks.8. The nanoparticle of any prior claim, wherein the nanoparticle furthercomprises a magnetic or magnezable disk that can be attracted to amagnet.
 9. The nanoparticle of claim 8, wherein the magnetic ormagnezable disk has a thickness in the range of 5 to 50 nm.
 10. Thenanoparticle of any prior claim, wherein the nanoparticle furthercomprises at least one metallic nano-dot on the edge of the spacerand/or the metallic disk.
 11. The nanoparticle of claim 10, wherein thenano-dots have a diameter in the range of 5 nm to 15 nm.
 12. Thenanoparticle of any prior claim, wherein the disks have the shapeselected from round, polygonal, pyramidal, elliptical, elongated barshaped, or any combination thereof.
 13. The nanoparticle of any priorclaim, wherein the metallic and the spacer have the same and similarlateral dimension.
 14. The nanoparticle of any prior claim, wherein, forlight enhancement in 800 nm wavelength and near by region, the diskshave a significantly round shape of diameter from 30 nm to 100 nm, thetop metallic disk thickness is from 5 nm to 30 nm, the spacer thicknessis from 2 to 30 nm, and the bottom metallic disk thickness is from 5 nmto 30 nm.
 15. The nanoparticle of any prior claim, wherein thenanoparticle further comprises a magnetic or magnezible disk, that canbe attracted to a magnet.
 16. The nanoparticle of any prior claim,wherein the stacked metallic disks are made of the same or differentmaterials.
 17. The nanoparticle of any prior claim, wherein the materialfor the metallic disks is selected from the group consisting of gold,silver, copper, aluminum, alloys thereof, and combinations thereof. 18.The nanodevice of any prior claim, wherein the distance between the pairof the metallic disk is in the range of 0.1 nm to 20 nm, for the lightwavelength of 800 nm and around.
 19. The nanodevice of any prior claim,wherein the lateral dimension of said metallic disc is in the range from5 nm to 150 nm.
 20. The nanodevice of any prior claim, wherein saidmetallic disc and the metallic back plane are spaced by a distance inthe range of 0.1 nm to 60 nm.
 21. The nanodevice of any prior claim,wherein said at least one metallic dot structure has dimensions in therange of 1 nm to 25 nm.
 22. The nanodevice of any prior claim, whereinthe distance between said metallic dot structure and said metallic disc,and the distance between said metallic dot structure and said metallicbackplane is in the range of 0.5 nm to 50 nm.
 23. A method of making afree-standing nanoparticle, comprising: (a) obtaining a templatecomprising a plurality of pillars on the surface, wherein the height ofthe pillar is greater than the thickness of the material to bedeposited; (b) depositing one of more materials on the top surface withthe pillars using a beam of the materials, in the directionsubstantially normal to the surface of the template; (c) separating thedeposited materials on top of the pillars from the pillars, therebyproducing said nanoparticles.
 24. The method of claim 23, wherein thepillars have a top area of the shape selected from round, polygonal,pyramidal, elliptical, elongated bar shaped, or any combination thereof.25. The method of claim 23 or 24, wherein the method comprises a step ofdepositing a magnetic or magnetizable layer on the stack produced instep b).
 26. An assay for analyte detection, comprising: (a) bringing ananalyte into proximity to or in contact with a free-standingnanoparticle comprising a pair of stacked metallic disks separated by anon-metallic spacer; (b) illuminating free-standing nanoparticle withlight that has a wavelength that is larger than the dimensions of thedisks and spacer; (c) detecting an outgoing signal from said analyteand/or free-standing nanoparticle.
 27. The assay of claim 26, whereinthe signal is selected from the group consisting of light absorption,light scattering, light reflection, and light radiation.
 28. The assayof claim 26 or 27, wherein the detecting is done by detecting Ramanscattering, color production, and/or luminescence that includesfluorescence, electroluminescence, chemiluminescence, andelectrochemiluminescence.
 29. The assay of any of claims 26-28, whereinthe analyte is bound by a capture agent that is on the surface of thenanoparticle.
 30. The assay of any of claims 26-29, wherein the analyteis selected from protein, peptides, DNA, RNA, nucleic acid, a smallmolecule, cell, and nanoparticle with different shape.